Methods and compositions based on diphtheria toxin-interleukin-3 conjugates

ABSTRACT

The present invention provides methods for inhibiting interleukin-3 receptor-expressing cells, and, in particular, inhibiting the growth of such cells by using a diphtheria toxin-human interleukin-3 conjugate (DT-IL3) that is toxic to cells expressing the interleukin-3 receptor. In preferred embodiments, the DT-IL3 conjugate is a fusion protein comprising amino acids 1-388 of diphtheria toxin fused via a peptide linker to full-length, human interleukin-3. In certain embodiments, the methods of the present invention relate to the administration of a DT-IL3 conjugate to inhibit the growth of cancer cells and/or cancer stem cells in humans, which cells express one or more subunits of the interleukin-3 receptor. Exemplary cells include myeloid leukemia cancer stem cells. In other embodiments, the methods of the present invention relate to ex vivo purging of bone marrow or peripheral blood to remove cells that express one or more subunits of the interleukin-3 receptor such that the purged bone marrow or peripheral blood is suitable, e.g., for autologous stem cell transplantation to restore hematopoietic function.

This application is a continuation of U.S. application Ser. No.12/368,048, filed Feb. 9, 2009, which is a continuation of U.S.application Ser. No. 11/899,747, filed Sep. 7, 2007, now U.S. Pat. No.7,763,242, which claims and is entitled to priority benefit of U.S.provisional application No. 60/843,471, filed Sep. 7, 2006 and U.S.provisional application No. 60/932,772, filed Jun. 1, 2007, all of whichare incorporated herein by reference in their entirety.

1. FIELD OF THE INVENTION

The present invention provides methods for targeting interleukin-3receptor-expressing cells, and, in particular, inhibiting the growth ofsuch cells by using a diphtheria toxin-human interleukin-3 conjugate(DT-IL3) that is toxic to cells expressing the interleukin-3 receptor.In preferred embodiments, the DT-IL3 conjugate is a recombinantconstruct wherein DNA encoding IL-3 is inserted in place of the receptorbinding domain of the diphtheria toxin (the catalytic and translocationregions of the diphtheria toxin remaining intact) that when translatedproduces a protein comprising amino acids 1-388 of diphtheria toxinfused via a peptide linker to full-length, human interleukin-3. Incertain embodiments, the methods of the present invention relate to theadministration of a DT-IL3 conjugate to inhibit the growth of cancercells and/or cancer stem cells in humans, which cells express one ormore subunits of the interleukin-3 receptor. Exemplary cells include thecancer cells and cancer stem cells of acute myeloid leukemia andmyelodysplastic syndrome. In other embodiments, the methods of thepresent invention relate to ex vivo purging of bone marrow or peripheralblood to remove cells that express one or more subunits of theinterleukin-3 receptor such that the purged bone marrow or peripheralblood is suitable, e.g., for autologous stem cell transplantation backinto the patient to restore hematopoietic function (e.g. as may berequired following high dose chemotherapy for cancer).

2. BACKGROUND OF THE INVENTION

2.1 Cancer Therapy

Cancer is one of the most significant health conditions. The AmericanCancer Society's Cancer Facts and Figures, 2003, predicts over 1.3million Americans will receive a cancer diagnosis this year. In theUnited States, cancer is second only to heart disease in mortalityaccounting for one of four deaths. In 2002, the National Institutes ofHealth estimated total costs of cancer totaled $171.6 billion, with $61billion in direct expenditures. The incidence of cancer is widelyexpected to increase as the US population ages, further augmenting theimpact of this condition. The current treatment regimens for cancerestablished in the 1970s and 1980s, have not changed dramatically. Thesetreatments, which include chemotherapy, radiation and other modalitiesincluding newer targeted therapies, have shown limited overall survivalbenefit when utilized in most advanced stage common cancers since, amongother things, these therapies primarily target tumor bulk.

More specifically, conventional cancer diagnosis and therapies to datehave attempted to selectively detect and eradicate neoplastic cells thatare largely fast-growing (i.e., cells that form the tumor bulk).Standard oncology regimens have often been largely designed toadminister the highest dose of irradiation or a chemotherapeutic agentwithout undue toxicity, i.e., often referred to as the “maximumtolerated dose” (MTD) or “no observed adverse effect level” (NOAEL).Many conventional cancer chemotherapies (e.g., alkylating agents such ascyclophosphamide, antimetabolites such as 5-Fluorouracil, and plantalkaloids such as vincristine) and conventional irradiation therapiesexert their toxic effects on cancer cells largely by interfering withcellular mechanisms involved in cell growth and DNA replication.Chemotherapy protocols also often involve administration of acombination of chemotherapeutic agents in an attempt to increase theefficacy of treatment. Despite the availability of a large variety ofchemotherapeutic agents, these therapies have many drawbacks (see, e.g.,Stockdale, 1998, “Principles Of Cancer Patient Management” in ScientificAmerican Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect.X). For example, chemotherapeutic agents are notoriously toxic due tonon-specific side effects on fast-growing cells whether normal ormalignant; e.g. chemotherapeutic agents cause significant, and oftendangerous, side effects, including bone marrow depression,immunosuppression, and gastrointestinal distress, etc.

Other types of traditional cancer therapies include surgery, hormonaltherapy, immunotherapy, anti-angiogenesis therapy, targeted therapy(e.g., therapy directed to a cancer target such as Gleevec® and othertyrosine kinase inhibitors, Velcade®, Sutent®, et al.), and radiationtreatment to eradicate neoplastic cells in a patient (see, e.g.,Stockdale, 1998, “Principles of Cancer Patient Management,” inScientific American: Medicine, vol. 3, Rubenstein and Federman, eds.,ch. 12, sect. IV). All of these approaches can pose significantdrawbacks for the patient including a lack of efficacy (in terms oflong-term outcome (e.g. due to failure to target cancer stem cells) andtoxicity (e.g. due to non-specific effects on normal tissues)).Accordingly, new therapies for improving the long-term prospect ofcancer patients are needed.

2.2 Cancer Stem Cells

Cancer stem cells comprise a unique subpopulation (often 0.1-10% or so)of a tumor that, relative to the remaining 90% or so of the tumor (i.e.,the tumor bulk), are more tumorigenic, relatively more slow-growing orquiescent, and often relatively more chemoresistant than the tumor bulk.Given that conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e. those cancer cellsthat comprise the tumor bulk), cancer stem cells which are oftenslow-growing may be relatively more resistant than faster growing tumorbulk to conventional therapies and regimens. Cancer stem cells canexpress other features which make them relatively chemoresistant such asmulti-drug resistance and anti-apoptotic pathways. The aforementionedwould constitute a key reason for the failure of standard oncologytreatment regimens to ensure long-term benefit in most patients withadvanced stage cancers—i.e. the failure to adequately target anderadicate cancer stem cells. In some instances, a cancer stem cell(s) isthe founder cell of a tumor (i.e., it is the progenitor of the cancercells that comprise the tumor bulk).

Cancer stem cells have been identified in a large variety of cancertypes. For instance, Bonnet et al., using flow cytometry were able toisolate the leukemia cells bearing the specific phenotype CD34+ CD38−,and subsequently demonstrate that it is these cells (comprising <1% of agiven leukemia), unlike the remaining 99+% of the leukemia bulk, thatare able to recapitulate the leukemia from whenst it was derived whentransferred into immunodeficient mice. See, e.g., “Human acute myeloidleukemia is organized as a hierarchy that originates from a primitivehematopoietic cell,” Nat. Med. 3:730-737 (1997). That is, these cancerstem cells were found as <1 in 10,000 leukemia cells yet this lowfrequency population was able to initiate and serially transfer a humanleukemia into severe combined immunodeficiency/non-obese diabetic(NOD/SCID) mice with the same histologic phenotype as in the originaltumor.

Cox et al. identified small subfractions of human acute lymphoblasticleukemia (ALL) cells which had the phenotypes CD34⁺/CD10⁻ andCD34⁺/CD19⁻, and were capable of engrafting ALL tumors inimmunocompromised mice—i.e. the cancer stem cells. In contrast, noengraftment of the mice was observed using the ALL bulk, despite, insome cases, injecting 10-fold more cells. See Cox et al.,“Characterization of acute lymphoblastic leukemia progenitor cells,”Blood 104(19): 2919-2925 (2004).

Multiple myeloma was found to contain small subpopulations of cells thatwere CD138− and, relative to the large bulk population of CD138+ myelomacells, had greater clonogenic and tumorigenic potential. See Matsui etal., “Characterization of clonogenic multiple myeloma cells,” Blood103(6): 2332. The authors concluded that the CD138− subpopulation ofmultiple myeloma was the cancer stem cell population.

Kondo et al. isolated a small population of cells from a C6-glioma cellline, which was identified as the cancer stem cell population by virtueof its ability to self-renew and recapitulate gliomas inimmunocompromised mice. See Kondo et al., “Persistence of a smallpopulation of cancer stem-like cells in the C6 glioma cell line,” Proc.Natl. Acad. Sci. USA 101:781-786 (2004). In this study, Kondo et al.determined that cancer cell lines contain a population of cancer stemcells that confer the ability of the line to engraft immunodeficientmice.

Breast cancers were shown to contain a small population of cells withstem cell characteristics (bearing surface markersCD44+CD24^(low lin−)). See Al-Hajj et al., “Prospective identificationof tumorigenic breast cancer cells,” Proc. Natl. Acad. Sci. USA100:3983-3988 (2003). As few as 200 of these cells, corresponding to1-10% of the total tumor cell population, are able to form tumors inNOD/SCID mice. In contrast, implantation of 20,000 cells that lackedthis phenotype (i.e. the tumor bulk) was unable to re-grow the tumor.

A subpopulation of cells derived from human prostate tumors was found toself-renew and to recapitulate the phenotype of the prostate tumor fromwhich they were derived thereby constituting the prostate cancer stemcell population. See Collins et al., “Prospective Identification ofTumorigenic Prostate Cancer Stem Cells,” Cancer Res 65(23):10946-10951(2005).

Fang et al. isolated a subpopulation of cells from melanoma with cancerstem cell properties. In particular, this subpopulation of cells coulddifferentiate and self-renew. In culture, the subpopulation formedspheres whereas the more differentiated cell fraction from the lesionswere more adherent. Moreover, the subpopulation containing sphere-likecells were more tumorigenic than the adherent cells when grafted intomice. See Fang et al., “A Tumorigenic Subpopulation with Stem CellProperties in Melanomas,” Cancer Res 65(20): 9328-9337 (2005).

Singh et al. identified brain tumor stem cells. When isolated andtransplanted into nude mice, the CD133+ cancer stem cells, unlike theCD133− tumor bulk cells, form tumors that can then be seriallytransplanted. See Singh et al., “Identification of human brain tumorinitiating cells,” Nature 432:396-401 (2004); Singh et al., “Cancer stemcells in nervous system tumors,” Oncogene 23:7267-7273 (2004); Singh etal., “Identification of a cancer stem cell in human brain tumors,”Cancer Res. 63:5821-5828 (2003).

Since conventional cancer therapies target rapidly proliferating cells(i.e., cells that form the tumor bulk) these treatments are believed tobe relatively ineffective at targeting and impairing cancer stem cells.In fact, cancer stem cells, including leukemia stem cells, have indeedbeen shown to be relatively resistant to conventional chemotherapeutictherapies (e.g. Ara-C, daunorubicin) as well as newer targeted therapies(e.g. Gleevec®, Velcade®). Examples of cancer stem cells from varioustumors that are resistant to chemotherapy, and the mechanism by whichthey are resistant, are described in Table 1 below.

TABLE 1 CSC Type Resistance Mechanism Reference AML Ara-C QuiescenceGuzman. Blood ′01 AML Daunorubicin Drug Efflux, Anti- Costello. Cancerapoptosis Res ′00 AML Daunorubicin, Drug Efflux Wulf. Blood ′01mitoxantrone AML Quiescence Guan. Blood ′03 AML, MDS Anti-apoptosisSuarez. Clin Cancer Res ′04 CML Quiescence Holyoake. Blood ′99 CMLGleevec ® Quiescence Graham. Blood ′02 Myeloma Velcade ® Matsui. ASH 04For example, leukemic stem cells are relatively slow-growing orquiescent, express multi-drug resistance genes, and utilize otheranti-apoptotic mechanisms—features which contribute to theirchemoresistance. See Jordan et al., “Targeting the most critical cells:approaching leukemia therapy as a problem in stem cell biology,” Nat.Clin. Pract. Oncol. 2: 224-225 (2005). Further, cancer stem cells byvirtue of their chemoresistance may contribute to treatment failure, andmay also persist in a patient after clinical remission and theseremaining cancer stem cells may therefore contribute to relapse at alater date. See Behbood et al., “Will cancer stem cells provide newtherapeutic targets?” Carcinogenesis 26(4): 703-711 (2004). Therefore,targeting cancer stem cells is expected to provide for improvedlong-term outcomes for cancer patients. Accordingly, new therapeuticagents and/or regimens designed to target cancer stem cells are neededto reach this goal.2.3 Acute Myeloid Leukemia

Approximately forty thousand patients per year develop acute myeloidleukemia (AML) in the U.S., Canada, and Europe. See, e.g., Jamal et al.,Cancer Statisitics 56:106-130 (2006). AML is the most common leukemia inadults and the second most common leukemia in children. The prolongedhospitalizations associated with treatment and complications represent asignificant share of health care costs in these regions. Further, evenwith combination induction and consolidation chemotherapy, most patientsultimately relapse and die from their disease or complications oftreatment. See, e.g., Brune et al., “Improved leukemia-free survivalafter post-consolidation immunotherapy with histamine dihydrochlorideand interleukin-2 in acute myeloid leukemia: results of a randomizedphase III trial,” Blood 108(1):88-96 (2006). Novel therapies areurgently needed. Selective targeting of AML cells stem cells may providea safe and more effective therapy.

2.4 Myelodysplastic Syndrome

There are approximately 20,000 new cases of myelodysplastic syndrome(MDS) each year in the U.S. Patients with myelodysplastic syndromestypically have low blood cell counts in at least one or more of redblood cells, white blood cells, and platelets. Upon examination, thebone marrow usually is found to be dysplastic or hyperplastic, meaningthere are too many poorly functioning blood stem cells in the marrow. Asmall percentage of MDS patients have hypoplastic bone marrow, meaningthere are too few blood stem cells in the marrow, which make the diseaselook similar to aplastic anemia. Nearly half of people with MDS have nosymptoms at time of diagnosis. When signs and symptoms do occur they caninclude anemia, weakness, fatigue, headache, bruising, increasedbleeding, rash, fevers, mouth sores and lingering illness. MDS occurs atan increasing frequency in older people, but it can occur in childrentoo. In less than a third of patients, MDS progresses over time tobecome acute leukemia. The average age of diagnosis is 70 years old.Treatments for MDS may vary considerably, depending on the type of MDS,the history of the patient, and the age and ability to tolerate certaintreatment regimens. Treatment options include supportive care,chemotherapy-related agents, and stem cell transplantation (which istypically used only in patients under 50). However, the remission ratefor existing treatments in relatively low, and new therapies are needed.

2.5 Interleukin-3

Interleukin-3 (IL-3) is a cytokine that supports the proliferation anddifferentiation of multi-potential and committed myeloid and lymphoidprogenitors. See, e.g., Nitsche et al. “Interleukin-3 promotesproliferation and differentiation of human hematopoietic stem cells butreduces their repopulation potential in NOD/SCID mice,” Stem Cells 21:236-244 (2003). Human interleukin-3 mediates its effects by binding tohuman IL-3 receptor, which is a hetrodimeric structure and consists ofan IL-3 binding α-subunit and a β-subunit. The α subunit is essentialfor ligand binding and confers specificity on the receptor. The βsubunit is also shared by the granulocyte macrophage-colony stimulatingfactor (GM-CSF) and IL-5 receptors, and is required for high affinityligand binding and signal transduction. Binding of IL-3 induces theheterodimerization of the α- and β-receptor subunits. The IL-3 receptoris over-expressed, relative to certain normal hematopoietic cells, onmultiple hematological cancers including AML, B cell acute lymphocyticleukemia (B-ALL), hairy cell leukemia, Hodgkin's disease, and certainaggressive non-Hodgkin's lymphomas (Munoz. Haematologica 86:1261-1269,2001; Riccioni. Leuk Lymphoma 46:303-311, 2005; Testa. Leukemia18:219-226, 2004), as well as on the cancer stem cells of AML,myelodsyplastic syndrome (MDS), T cell ALL (T-ALL), and chronic myeloidleukemia (CML) (See Jordan et al. “The interleukin-3 receptor alphachain is a unique marker for human acute myelogenous leukemia stemcells,” Leukemia 14:1777-1784 (2000); Florian et al. “Detection ofmolecular targets on the surface of CD34+/CD38− stem cells in variousmyeloid malignancies,” Leuk. Lymphoma 47:207-222 (2006); Lhermitte etal. “Most immature T-ALLs express Ra-IL3 (CD123): possible target forDT-IL3# therapy,” 20:1908-1910 (2006); and Hogge et al. “VariantDiphtheria Toxin-Interleukin-3 Conjugates with Increased ReceptorAffinity Have Enhanced Cytotoxicity against Acute Myeloid LeukemiaProgenitors,” Clin. Caner Res. 12:1284-1291 (2004).

2.6 Diphtheria Toxin

Diphtheria toxin (DT) is a 535 amino acid protein with three domainsconsisting of a catalytic domain (amino acids 1-186) connected by anarginine-rich disulfide loop to a translocation domain (amino acids187-388) and a cell binding domain (amino acids 389-535; FIG. 1). See,e.g., Choe et al., “The crystal structure of diphtheria toxin,” Nature357: 216-222 (1992). Native DT binds to heparin-binding epidermal growthfactor precursor and CD9 on the cell surface, undergoes clathrin-,dynamin- and ATP-dependent receptor-mediated endocytosis and, withendosome acidification by vesicular ATPase, the DT translocation domainundergoes protonation of acidic residues and spontaneous insertion intothe vesicular membrane to form 18-22 Angstrom channels. The catalyticdomain unfolds and is cleaved by furin in the vesicle and then theC-terminus of the catalytic domain transfers through the channel andbinds to coatomer proteins, specifically β-COP. Protein disulfideisomerase reduces the linkage of the catalytic domain with the remainderof DT and the peptide passes into the cytosol. Hsp90 assists inrefolding. The DT fragment then ADP-ribosylates elongation factor 2leading to protein synthesis inactivation and cell death (FIG. 2). SeeRatts et al., “A conserved motif in transmembrane helix 1 of diphtheriatoxin mediates catalytic domain delivery to the cytosol,” Proc. Natl.Acad. Sci. 102: 15635-15640 (2005).

2.7 Recombinant Diphtheria Toxin Conjugates

Recombinant protein-toxin conjugates represent a novel class of oncologybiological agents that specifically target receptors on the surfaces ofcancer cells. These agents typically consist of a truncated toxin, oftenincluding the catalytic and translocation (but not cell binding)domains, fused to a cell selective ligand which directs the toxin to theintended target. One such technology involves the recombinant diphtheriatoxin (DT). DT is a 535 amino acid protein with three domains consistingof a catalytic domain (amino acids 1-186) connected by an arginine-richdisulfide loop to a translocation domain (amino acids 187-388) and acell binding domain (amino acids 389-535; FIG. 1). See, e.g., Choe etal., “The crystal structure of diphtheria toxin”, Nature. 357: 216-222(1992). Native DT binds to heparin-binding epidermal growth factorprecursor and CD9 on the cell surface, undergoes clathrin-, dynamin- andATP-dependent receptor-mediated endocytosis, and, with endosomeacidification by vesicular ATPase, the DT translocation domain undergoesprotonation of acidic residues and spontaneous insertion into thevesicular membrane to form 18-22 Angstrom channels. The catalytic domainunfolds and is cleaved by furin in the vesicle and then the C-terminusof the catalytic domain transfers through the channel and binds β-COP.Protein disulfide isomerase reduces the linkage of the catalytic domainwith the remainder of DT and the peptide passes into the cytosol. Hsp90assists in refolding. The DT fragment then ADP-ribosylates elongationfactor 2 leading to protein synthesis inactivation and cell death (FIG.2). See Ratts et al., “A conserved motif in transmembrane helix 1 ofdiphtheria toxin mediates catalytic domain delivery to the cytosol” ProcNatl Acad Sci., 102: 15635-15640 (2005). A number of recombinant DTconjugates, utilizing a truncated form of DT, have been expressed,purified, and tested in cell culture and selective cell toxicity hasbeen shown. One such recombinant toxin is the DT₃₈₈IL-3 conjugate,wherein the truncated DT maintains its catalytic and translocation, butnot its cell binding domain.

DT₃₈₈IL-3 was constructed by fusing the gene encoding the catalytic andtranslocation domains of DT (amino acids 1-388) via a Met-His linkerwith human IL-3. See, e.g., Frankel et al., “Diphtheria toxin fused tohuman interleukin-3 is toxic to blasts from patients with myeloidleukemias,” Leukemia 14: 576-585 (2000). DT₃₈₈IL-3 has been shown to bepotently and selectively cytotoxic to IL-3R positive AML cell lines andprimary leukemia cells derived from patients. (See, Frankel et al.,“Characterization of diphtheria fusion proteins targeted to the humaninterleukin-3 receptor,” Protein Eng. 13: 575-581 (2000); Alexander etal., “High affinity interleukin-3 receptor expression on blasts frompatients with acute myelogenous leukemia correlates with cytotoxicity ofa diphtheria toxin/IL-3 fusion protein,” Leuk. Res. 25: 875-881 (2001);Alexander et al. “In vitro interleukin-3 binding to leukemia cellspredicts cytotoxicity of a diphtheria toxin/IL-3 fusion protein,”Bioconj. Chem. 11:564-568 (2000); Feuring-Buske et al. “A diphtheriatoxin interleukin-3 fusion protein is cytotoxic to primitive acutemyeloid leukemia progenitors but spares normal progenitors,” Cancer Res.62: 1730-1736 (2002)). Additional studies found that high affinityvariants of the DT₃₈₈IL-3 compound, named DT388-IL3[K116W] (based on themutation of a lysine at amino acid 116 to tryptophan) andDT388IL3[Δ125-133] (based on a deletion of amino acids 125-133 in theIL3 domain), had increased potency against leukemia cells (See, Hogge etal., “Variant diphtheria toxin-interleukin-3 conjugates with increasedreceptor affinity have enhanced cytotoxicity against acute myeloidleukemia progenitors,” Clin. Cancer Res. 12: 1284-1291 (2006); Testa etal., “Diphtheria toxin fused to variant human interleukin-3 inducescytotoxicity of blasts from patients with acute myeloid leukemiaaccording to the level of interleukin-3 receptor expression,” Blood 106:2527-2529 (2005)). DT₃₈₈IL-3 also demonstrated in vivo anti-tumorefficacy in certain mouse models of human leukemia (See, Black et al.,“Diphtheria toxin interleukin-3 fusion protein (DT₃₈₈IL-3) prolongsdisease-free survival of leukemic immuno-compromised mice,” Leukemia;17: 155-159 (2003); Feuring-Buske et al. “A diphtheriatoxin-interleukin-3 fusion protein is cytotoxic to primitive acutemyeloid leukemia progenitors but spares normal progenitors,” Cancer Res.62: 1730-1736 (2002); and Hogge et al., “The efficacy ofdiphtheria-growth factor fusion proteins is enhanced byco-administration of cytosine arabinoside in an immunodeficient mousemodel of human acute myeloid leukemia” Leuk Res 28: 1221-1226 (2004)).Safety was shown at therapeutically active doses in rodents and monkeys.(See, Black et al., “Diphtheria toxin interleukin-3 fusion protein(DT₃₈₈IL-3) prolongs disease-free survival of leukemicimmuno-compromised mice,” Leukemia; 17: 155-159 (2003); Cohen et al.,“Toxicology and pharmacokinetics of DT₃₈₈IL-3, a fusion toxin consistingof a truncated diphtheria toxin (DT₃₈₈) linked to human interleukin-3(IL-3), in cynomolgus monkeys” Leuk Lymph, 45: 1647-1656 (2004); Cohenet al., “Safety evaluation of DT₃₈₈IL-3, a diphtheriatoxin-interleukin-3 fusion protein, in cynomolgus monkeys,” CancerImmunol. Immunother. 54: 799-806 (2005)). Clinical batches of DT₃₈₈IL-3were prepared and an IND obtained (BB IND #11314). (See, Urieto et al.,“Expression and purification of the recombinant diphtheria fusion toxinDT₃₈₈IL-3 for phase I clinical trials,” Protein Exp. Purif. 33: 123-133(2004).

3. SUMMARY OF THE INVENTION

The present invention is directed to a method for inhibitinginterleukin-3 receptor-expressing cells comprising administering to ahuman in need of such inhibition a pharmaceutical composition comprisingan amount of a human interleukin-3-diphtheria toxin conjugate effectivein inhibiting said cells and a pharmaceutically acceptable carrier, withthe proviso that the interleukin-3 receptor expressing cells are notacute myeloid leukemia cells, and wherein the cells express the alphaand beta subunits of the interleukin-3 receptor. In a preferred aspectof this embodiment, the growth of interleukin-3 receptor-expressingcells is inhibited.

In this or any of the embodiments of the present invention, theinterleukin-3-diphtheria toxin conjugate can comprise the full-length,mature (lacking the signal peptide), human interleukin-3 connected by acovalent bond to diphtheria toxin. Preferably, the diphtheria toxin ismodified in that the cell surface binding domain is deleted. Forexample, the conjugate is a chemical conjugate in which the diphtheriatoxin portion (the catalytic and translocation domains of diphtheriatoxin) and the interleukin-3 portion are chemically linked togethereither directly or through a chemical linker. Optionally, the conjugateis a genetic recombinant in which the conjugate is expressed as a singlepolypeptide. When the conjugate is a recombinant conjugate, thetranslated conjugate preferably comprises the catalytic andtranslocation domains of diphtheria toxin linked via a peptide bond tohuman interleukin-3 Most preferably, the conjugate comprises amino acids1-388 of diphtheria toxin linked via a peptide linker to humaninterleukin-3.

In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. In a specific aspect, theconjugate can be administered at or below a dose that is the maximumdose tolerated without undue toxicity. Further, the conjugate can beadministered at least two times a week or the conjugate can beadministered at least three times a week, at least four times a week, atleast five times a week, at least six times a week, or seven times aweek. In a specific aspect, where the conjugate is administered morethan once, the conjugate can be administered at a dose of 4 μg/kg perday or greater each time. In particular, the conjugate can beadministered over a period of one or two weeks or greater. In aspectswhere the growth of interleukin-3 receptor-expressing cells isinhibited, the growth of the cells can be inhibited by at least 50%, atleast 65%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or by at least 99% as compared to a reference sample, i.e., asample of cells not contacted with a conjugate of the invention.

In another embodiment, the present invention is directed to a method forinhibiting the growth of interleukin-3 receptor-expressing cellscomprising administering to a human in need of such inhibition apharmaceutical composition comprising an amount of aninterleukin-3-diphtheria toxin conjugate effective in inhibiting saidcells and a pharmaceutically acceptable carrier, in which the conjugateis administered at a dose greater than 4 μg/kg per day, and wherein thecells express the alpha subunit of the interleukin-3 receptor. In anaspect of this embodiment, the cells express both the alpha and the betasubunits of the interleukin-3 receptor.

In specific aspects of this embodiment, the conjugate can beadministered at a dose in a range of greater than 4 μg/kg per day toabout 20 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of greater than 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect, the conjugate can be administeredat or below a dose that is the maximum dose tolerated without unduetoxicity. Further, the conjugate can be administered at least two timesa week or the conjugate can be administered at least three times a week,at least four times a week, at least five times a week, at least sixtimes a week, or seven times a week. In a specific aspect, where theconjugate is administered more than once, the conjugate can beadministered at a dose of greater than 4 μg/kg per day each time. Inparticular, the conjugate can be administered over a period of two weeksor greater. In certain aspects, the growth of interleukin-3 receptorexpressing cells can be inhibited by at least 50%, at least 65%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or byat least 99% as compared to a reference sample, i.e., a sample of cellsnot contacted with a conjugate of the invention. In a specific aspect ofthis embodiment, the conjugate can be administered at a dose of about5.3 μg/kg per day, or at a dose of about 7.1 μg/kg per day, or at a doseof about 9.4 μg/kg per day, or at a dose of about 12.5 μg/kg per day.

In yet another embodiment, the present invention is directed to a methodof treating, preventing and/or managing a disease or disorder thatdisplays or is characterized by interleukin-3 receptor-expressioncomprising administering to a human in need of such treatment orprevention a pharmaceutical composition comprising an amount of aninterleukin-3-diphtheria toxin conjugate effective to treat, prevent ormanage the disease or disorder and a pharmaceutically acceptablecarrier, with the proviso that the disease or disorder is not acutemyeloid leukemia, and wherein the cells express the alpha and betasubunits of the interleukin-3 receptor. In one aspect of thisembodiment, interleukin-3 receptor expression can be overexpression ofone or more subunits of the interleukin-3 receptor on cells thatnormally express the interleukin-3 receptor. In another aspect,interleukin-3 receptor expression can be inappropriate expression ofinterleukin-3 receptor on cells that do not normally express one or moresubunits of the interleukin-3 receptor. In yet another aspect, thedisease or disorder that displays or is characterized by the presence orover-presence of a type of cell that expresses one or more subunits ofthe interleukin-3 receptor. Exemplary diseases or disorders that can betreated in this embodiment of the invention include, but are not limitedto, allergic diseases or disorders, autoimmune diseases or disorders,inflammatory diseases or disorders, or cancers that are not acutemyeloid leukemia. In aspects where the disease or disorder is cancer,the cancer can be refractory, or multidrug resistant. In someembodiments, the disease or disorder is MDS.

In still another embodiment, the present invention is directed to amethod of treating, preventing and/or managing a disease or disorderthat displays or is characterized by interleukin-3 receptor-expressioncomprising administering to a human in need of such treatment,prevention and/or management a pharmaceutical composition comprising anamount of an interleukin-3-diphtheria toxin conjugate effective totreat, prevent and/or manage the disease or disorder and apharmaceutically acceptable carrier, wherein the cells express the alphasubunit of the interleukin-3 receptor. In one aspect of this embodiment,the cells express both the alpha and beta subunits of the interleukin-3receptor. In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. In a specific aspect, theconjugate can be administered at or below a dose that is the maximumdose tolerated without undue toxicity. In one aspect of this embodiment,interleukin-3 receptor expression can be overexpression of interleukin-3receptor on cells that normally express the interleukin-3 receptor. Inanother aspect, interleukin-3 receptor expression can be inappropriateexpression of interleukin-3 receptor on cells that do not normallyexpress the interleukin-3 receptor. In yet another aspect, the diseaseor disorder that displays or is characterized by the presence orover-presence of a type of cell that expresses the interleukin-3receptor. Exemplary diseases or disorders that can be treated in thisembodiment of the invention include, but are not limited to, allergicdiseases or disorders, autoimmune diseases or disorders, inflammatorydiseases or disorders, or cancers (including without limitation acutemyeloid leukemia). In aspects where the disease or disorder is cancer,the cancer can be refractory, or multidrug resistant. In someembodiments, the disease or disorder is MDS.

In yet another embodiment of the present invention, a method fortreating, preventing, and/or managing cancer is provided, which methodcomprises administering to a human in need of such treatment, preventionand/or management a pharmaceutical composition comprising an amount ofan interleukin-3-diphtheria toxin conjugate effective to treat, preventand/or manage the cancer and a pharmaceutically acceptable carrier,wherein the cancer cells express the alpha and beta subunits of theinterleukin-3 receptor, with the proviso that the cancer is not acutemyeloid leukemia. In yet another embodiment, the present invention isdirected to a method for treating or preventing cancer, comprisingadministering to a human in need of such treatment or prevention apharmaceutical composition comprising an amount of aninterleukin-3-diphtheria toxin conjugate effective to treat or preventthe cancer and a pharmaceutically acceptable carrier, wherein the cancerstem cells express the alpha and beta subunits of the interleukin-3receptor, with the proviso that the cancer is not acute myeloidleukemia.

In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. In a specific aspect, theconjugate can be administered at or below a dose that is the maximumdose tolerated without undue toxicity. Further, the conjugate can beadministered at least two times a week or the conjugate can beadministered at least three times a week, at least four times a week, atleast five times a week, at least six times a week, or seven times aweek. In a specific aspect, where the conjugate is administered morethan once, the conjugate can be administered at a dose of 4 μg/kg perday or greater each time. In particular, the conjugate can beadministered over a period of two weeks or greater. In other aspects,the growth of the cancer cells or the cancer stem cells can be inhibitedby at least 50%, at least 65%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95% or by at least 99% as compared to a referencesample, i.e., a sample of cells not contacted with a conjugate of theinvention.

In other aspects of these embodiments, the human patient can be in astate of remission from the cancer. In yet other aspects, the humanpatient has been previously treated with the conjugate or has beenpreviously treated with conventional chemotherapeutic agents or hasradiation therapy. In yet another aspect, the human patient, concurrentwith treatment with compounds of the invention, can be administered aconventional chemotherapeutic agent or can undergo radiation therapy. Inother aspects, the human patient has no detectable levels ofanti-diphtheria toxin antibodies prior to administration of a conjugateof the invention. In yet another aspect, the method further comprisesadministering a conventional chemotherapeutic agent. In particularaspects, the cancer is a non-hematologic cancer. Further the cancer canbe refractory or multi-drug resistant.

In a specific aspect, the methods of this embodiment can furthercomprise monitoring the amount of cancer cells or cancer stem cellsexpressing the alpha subunit (in some embodiments, the alpha and betasubunits) of the interleukin-3 receptor in a sample derived from thehuman after administration of a conjugate of the invention anddetermining a further course of treatment based on the amount of cancercells or cancer stem cells expressing the alpha subunit (in someembodiments, the alpha and beta subunits) present in the sample ascompared to a reference sample or a sample of cancer cells or cancerstem cells obtained from the human before or during administration ofthe conjugate.

In yet another embodiment, the present invention is directed to a methodfor treating, preventing, and/or managing myeloid leukemia comprisingadministering to a human in need of such treatment, prevention, and/ormanagement a pharmaceutical composition comprising an amount of aninterleukin-3-diphtheria toxin conjugate effective to treat or preventmyeloid leukemia and a pharmaceutically acceptable carrier, in which theconjugate is administered at a dose of greater than 4 μg/kg, and whereinthe myeloid leukemia cells express the alpha and beta subunits of theinterleukin-3 receptor. In particular, the myeloid leukemia can be acutemyeloid leukemia, chronic myeloid leukemia, or myelodysplastic syndrome.In some cases the myeloid leukemia may be refractory and/or multi-drugresistant. In certain aspects of this embodiment, the myeloid leukemiacells can express both the alpha and the beta subunits of theinterleukin-3 receptor.

In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. Further, the conjugate can beadministered at least two times a week or the conjugate can beadministered at least three times a week, at least four times a week, atleast five times a week, at least six times a week, or seven times aweek. In a specific aspect, where the conjugate is administered morethan once, the conjugate can be administered at a dose of greater than 4μg/kg per day each time. In particular, the conjugate can beadministered over a period of two weeks or greater. In certain aspects,the amount of myeloid leukemia cells can be decreased by at least 50%,at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or by at least 99% as compared to a reference sample, i.e., asample of cells not contacted with a conjugate of the invention. In aspecific aspect of this embodiment, the conjugate can be administered ata dose of about 5.3 μg/kg per day, or at a dose of about 7.1 μg/kg perday, or at a dose of about 9 μg/kg per day.

In other aspects of these embodiments, the human patient can be in astate of remission of the myeloid leukemia. In yet other aspects, thehuman patient has been previously treated with the conjugate or has beenpreviously treated with conventional chemotherapeutic agents orradiation therapy. In yet another aspect, the human patient concurrentlycan be administered a conventional chemotherapeutic agent or radiationtherapy. In yet another aspect, the human patient is administered theconjugate 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 9 months, or 12 months after receiving conventionalchemotherapy. In other aspects, the human patient has low levels or nodetectable levels of anti-diphtheria toxin antibodies prior toadministration of a conjugate of the invention. In yet another aspect,the method further comprises administering a conventionalchemotherapeutic agent.

In a specific aspect, the methods of this embodiment can furthercomprise monitoring the amount of myeloid leukemia cells expressing thealpha and/or beta subunits of the interleukin-3 receptor in a samplederived from the human after administration of a conjugate of theinvention and determining a further course of treatment based on theamount of myeloid leukemia cells expressing the alpha and/or betasubunits present in the sample as compared to a reference sample or asample of myeloid leukemia cells obtained from the human before orduring administration of the conjugate.

In a specific aspect, the methods of this embodiment can furthercomprise monitoring the amount of myeloid leukemia cells expressing thealpha subunit of the interleukin-3 receptor in a sample derived from thehuman after administration of a conjugate of the invention anddetermining a further course of treatment based on the amount of myeloidleukemia cells expressing the alpha subunit present in the sample ascompared to a reference sample or a sample of myeloid leukemia cellsobtained from the human before or during administration of theconjugate.

The present invention is also directed to a method for preventing arelapse of cancer in a human previously treated for the cancer,comprising administering to a human in need of such prevention who hadbeen previously treated for cancer, a pharmaceutical compositioncomprising an amount of an interleukin-3-diphtheria toxin conjugateeffective to prevent the relapse of the cancer and a pharmaceuticallyacceptable carrier, wherein the cancer cells or the cancer stem cellsexpress the alpha and beta subunits of the interleukin-3 receptor, withthe proviso that the cancer is not myeloid leukemia. In anotherembodiment, the invention is directed to a method for preventing arelapse of myeloid leukemia in a human previously treated for myeloidleukemia, comprising administering to a human in need of such preventionwho had been previously treated for myeloid leukemia, a pharmaceuticalcomposition comprising an amount of an interleukin-3-diphtheria toxinconjugate effective to prevent the relapse of myeloid leukemia and apharmaceutically acceptable carrier, in which the conjugate isadministered at a dose of greater than 4 μg/kg per day.

In yet another embodiment, a method for preventing a relapse of cancerin a human in remission from such cancer is provided, which methodcomprises administering to a human in need of such prevention who is inremission from said cancer, a pharmaceutical composition comprising anamount of an interleukin-3-diphtheria toxin conjugate effective toprevent the relapse of the cancer and a pharmaceutically acceptablecarrier, wherein the cancer cells or the cancer stem cells express thealpha and beta subunits of the interleukin-3 receptor, with the provisothat the cancer is not myeloid leukemia. Another embodiment of theinvention is directed to a method for preventing a relapse of myeloidleukemia in a human in remission from myeloid leukemia, comprisingadministering to a human in need of such prevention who is in remissionfrom myeloid leukemia, a pharmaceutical composition comprising an amountof an interleukin-3-diphtheria toxin conjugate effective to prevent therelapse of myeloid leukemia and a pharmaceutically acceptable carrier,in which the conjugate is administered at a dose of greater than 4μg/kg.

The present invention is also directed to a method for purging bonemarrow or peripheral blood prior to autologous stem cell transplant,comprising contacting ex vivo bone marrow or peripheral blood obtainedfrom a human with a composition comprising an amount of aninterleukin-3-diphtheria toxin conjugate for a time sufficient tosignificantly purge the bone marrow or peripheral blood of cellsexpressing the alpha and beta subunits of the interleukin-3 receptor. Inan aspect of this embodiment, the amount of bone marrow or peripheralblood cells expressing a beta subunit of the interleukin-3 receptorafter contacting with a conjugate of the invention can be decreased byat least 50%, 60%, 75%, 80%, 90%, 95%, or by at least 99%. The presentinvention is also directed to a method for performing an autologous bonemarrow or peripheral blood stem cell transplant, comprisingadministering to a human an amount of significantly purged bone marrowor peripheral blood effective to reconstitute hematopoietic function insaid human, wherein said purged bone marrow or peripheral blood is bonemarrow or peripheral blood obtained from said human previously contactedwith an amount of an interleukin-3-diphtheria toxin conjugate for a timesufficient to significantly purge the bone marrow or peripheral blood ofcells expressing the alpha and beta subunits of the interleukin-3receptor. Further, the present invention is directed to a compositioncomprising purged bone marrow or peripheral blood, wherein said purgedbone marrow or peripheral blood is bone marrow or peripheral bloodobtained from a human and contacted ex vivo with an amount of aninterleukin-3-diphtheria toxin conjugate for a time sufficient tosignificantly purge the bone marrow or peripheral blood of cellsexpressing the alpha and beta subunits of the interleukin-3 receptor,and then possibly re-introducing the bone marrow or peripheral bloodcells back into the patient. In one aspect, the composition can furthercomprise a pharmaceutically acceptable carrier.

In certain embodiments of the invention, conventional chemotherapy andthe methods of the invention may be used sequentially. In a specificaspect of this embodiment, the patient's leukemia blasts are firstreduced by use of conventional chemotherapy, followed by a regimencomprising administration of an amount of an interleukin-3-diphtheriatoxin conjugate for a time sufficient to significantly stabilize,reduce, or eradicate cancer stem cells expressing the alpha and betasubunits of the interleukin-3 receptor.

3.1 DEFINITIONS

As used herein, the term “agent” refers to any molecule, compound,and/or substance for use in the prevention, treatment, management and/ordiagnosis of cancer, including the diphtheria toxin-interleukin-3conjugate of the invention.

As used herein, the term “conjugate of the invention” refers tointerleukin-3 or a portion, analog or derivative thereof that binds tothe interleukin-3 receptor or subunit thereof conjugated to diphtheriatoxin, a portion thereof or an analog thereof. Unless otherwiseindicated, the terms “compound of the invention” and “composition of theinvention” are used as alternatives for the term “conjugate of theinvention.”

As used herein, the term “amount,” as used in the context of the amountof a particular cell population or cells, refers to the frequency,quantity, percentage, relative amount, or number of the particular cellpopulation or cells.

As used herein, the terms “about” or “approximately,” unless otherwiseindicated, refer to a value that is no more than 10% above or below thevalue being modified by the term.

As used herein, the term “significantly,” as used in the context ofpurging of the bone marrow or peripheral blood of cells expressing thealpha and beta subunits of the interleukin-3 receptor, refers to adecrease in cells expressing the alpha and beta subunits of theinterleukin-3 receptor by at least 50%, 60%, 75%, 80%, 90%, 95%, or 99%.

As used herein, the term “small reduction,” in the context of aparticular cell population (e.g., circulating endothelial cells and/orcirculating endothelial progenitors) refers to less than a 30% reductionin the cell population (e.g., the circulating endothelial cellpopulation and/or the circulating endothelial progenitor population).

As used herein, the phrase “diagnostic agent” refers to any molecule,compound, and/or substance that is used for the purpose of diagnosingcancer. Non-limiting examples of diagnostic agents include antibodies,antibody fragments, or other proteins, including those conjugated to adetectable agent. As used herein, the term “detectable agents” refer toany molecule, compound and/or substance that is detectable by anymethodology available to one of skill in the art. Non-limiting examplesof detectable agents include dyes, gases, metals, or radioisotopes. Asused herein, diagnostic agent and “imaging agent” are equivalent terms.

As used herein, the phrase “prophylactic agent” refers to any molecule,compound, and/or substance that is used for the purpose of preventingcancer. Examples of prophylactic agents include, but are not limited to,proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs,Ig fragments, polyclonal antibodies and their fragments, monoclonalantibodies and their fragments), binding proteins, chemospecific agents,chemotoxic agents (e.g., anti-cancer agents), proliferation basedtherapy, and small molecule drugs.

As used herein, the term “therapeutic agent” refers to any molecule,compound, and/or substance that is used for the purpose of treatingand/or managing a disease or disorder. Examples of therapeutic agentsinclude, but are not limited to, proteins, immunoglobulins (e.g.,multi-specific Igs, single chain Igs, Ig fragments, polyclonalantibodies and their fragments, monoclonal antibodies and theirfragments), peptides (e.g., peptide receptors, selectins), bindingproteins, biologics, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), proliferation-based therapy, radiation,chemotherapy, anti-angiogenic agents, and small molecule drugs.

As used herein, the term “proliferation based therapy” refers to anymolecule, compound, substance, and/or method that differentiallyimpairs, inhibits or kills rapidly proliferating cell populations (e.g.,cancer cells) in comparison with cell populations that divide moreslowly. Proliferation based therapies may include, but are not limitedto those chemotherapeutic and radiation therapies that are typicallyused in oncology. A proliferation based agent may differentially impair,inhibit or kill rapidly proliferating cells by any mechanism known toone skilled in the art including, but not limited to, disrupting DNAfunction (including DNA replication), interfering with enzymes involvedin DNA repair, intercalating DNA, interfering with RNA transcription ortranslation, interfering with enzymes involved with DNA replication,interfering with a topoisomerase, such as topoisomerase II, interferingwith mitosis, and inhibiting enzymes necessary for the synthesis ofproteins needed for cellular replication. Specific examples ofproliferation based therapies include, but are not limited to,alkylating agents, nitrosoureas, antimetabolites, antibiotics,procarbazine, hydroxyurea, platinum-based agents, anthracycline,topoisomerase II inhibitors, spindle poisons, and mitotic inhibitors.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. Non-limitingexamples include those cancers described in Section 5.3.2. The term“cancer” encompasses a disease involving both pre-malignant andmalignant cancer cells. In some embodiments, cancer refers to alocalized overgrowth of cells that has not spread to other parts of asubject, i.e., a localized, or at times benign, tumor. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Inyet other embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “cancer cells” refer to cells that acquire acharacteristic set of functional capabilities during their development,including the ability to evade apoptosis, self-sufficiency in growthsignals, insensitivity to anti-growth signals, tissueinvasion/metastasis, significant growth potential, and/or sustainedangiogenesis. The term “cancer cell” is meant to encompass bothpre-malignant and malignant cancer cells.

As used herein, the term “cancer stem cell(s)” refers to a cell that canbe a progenitor of a highly proliferative cancer cell. A cancer stemcell has the ability to re-grow a tumor as demonstrated by its abilityto form tumors in immunocompromised mice, and typically to form tumorsupon subsequent serial transplantation in immunocompromised mice. Cancerstem cells are also typically slow-growing relative to the bulk of atumor; that is, cancer stem cells are generally quiescent. In certainembodiments, but not all, the cancer stem cell may representapproximately 0.1 to 10% of a tumor.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of cancer and one or more symptomsthereof, to enhance or improve the prophylactic effect(s) of anothertherapy, reduce the severity, the duration of cancer, ameliorate one ormore symptoms of cancer, prevent the advancement of cancer, causeregression of cancer, and/or enhance or improve the therapeuticeffect(s) of another therapy. In an embodiment of the invention, theamount of a therapy is effective to achieve one, two, three, or moreresults following the administration of one, two, three or moretherapies: (1) a stabilization, reduction or elimination of the cancerstem cell population; (2) a stabilization, reduction or elimination inthe cancer cell population; (3) a stabilization or reduction in thegrowth of a tumor or neoplasm; (4) an impairment in the formation of atumor; (5) eradication, removal, or control of primary, regional and/ormetastatic cancer; (6) a reduction in mortality; (7) an increase indisease-free, relapse-free, progression-free, and/or overall survival,duration, or rate; (8) an increase in the response rate, the durabilityof response, or number of patients who respond or are in remission; (9)a decrease in hospitalization rate, (10) a decrease in hospitalizationlengths, (11) the size of the tumor is maintained and does not increaseor increases by less than 10%, preferably less than 5%, preferably lessthan 4%, preferably less than 2%, (12) an increase in the number ofpatients in remission, (13) an increase in the length or duration ofremission, (14) a decrease in the recurrence rate of cancer, (15) anincrease in the time to recurrence of cancer, and (16) an ameliorationof cancer-related symptoms and/or quality of life.

As used herein, the phrase “elderly human” refers to a human between 65years old or older, preferably 70 years old or older.

As used herein, the phrase “human adult” refers to a human 18 years ofage or older.

As used herein, the phrase “human child” refers to a human between 24months of age and 18 years of age.

As used herein, the phrase “human infant” refers to a human less than 24months of age, preferably less than 12 months of age, less than 6 monthsof age, less than 3 months of age, less than 2 months of age, or lessthan 1 month of age.

As used herein, the phrase “human patient” refers to any human, whetherelderly, an adult, child or infant.

As used herein, the term “refractory” is most often determined byfailure to reach clinical endpoint, e.g., response, extended duration ofresponse, extended disease free, survival, relapse free survival,progression free survival and overall survival. Another way to definebeing refractory to a therapy is that a patient has failed to achieve aresponse to a therapy such that the therapy is determined to not betherapeutically effective.

As used herein, the term “specifically binds to an antigen” andanalogous terms refer to peptides, polypeptides, proteins, fusionproteins and antibodies or fragments thereof that specifically bind toan antigen or a fragment and do not specifically bind to other antigens.A peptide, polypeptide, protein, or antibody that specifically binds toan antigen may bind to other peptides, polypeptides, or proteins withlower affinity as determined by, e.g., immunoassays, BIAcore, or otherassays known in the art. Antibodies or fragments that specifically bindto an antigen may be cross-reactive with related antigens. Preferably,antibodies or fragments that specifically bind to an antigen do notcross-react with other antigens. An antibody binds specifically to anantigen when it binds to the antigen with higher affinity than to anycross-reactive antigen as determined using experimental techniques, suchas radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays(ELISAs). See, e.g., Paul, ed., 1989, Fundamental Immunology, 2^(nd)ed., Raven Press, New York at pages 332-336 for a discussion regardingantibody specificity.

As used herein, the term “in combination” in the context of theadministration of a therapy to a subject refers to the use of more thanone therapy (e.g., prophylactic and/or therapeutic). The use of the term“in combination” does not restrict the order in which the therapies(e.g., a first and second therapy) are administered to a subject. Atherapy can be administered prior to (e.g., 1 minute, 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy to a subject which had, has, or is susceptible to cancer.The therapies are administered to a subject in a sequence and within atime interval such that the therapies can act together. In a particularembodiment, the therapies are administered to a subject in a sequenceand within a time interval such that they provide an increased benefitthan if they were administered otherwise. Any additional therapy can beadministered in any order with the other additional therapy.

As used herein, the terms “manage,” “managing,” and “management” in thecontext of the administration of a therapy to a subject refer to thebeneficial effects that a subject derives from a therapy (e.g., aprophylactic or therapeutic agent) or a combination of therapies, whilenot resulting in a cure of cancer. In certain embodiments, a subject isadministered one or more therapies (e.g., one or more prophylactic ortherapeutic agents) to “manage” cancer so as to prevent the progressionor worsening of the condition.

As used herein, the term “marker” in the context of a cell or tissue(e.g. a normal or cancer cell or tumor) means any antigen, molecule orother chemical or biological entity that is specifically found in or ona tissue that it is desired to identified or identified in or on aparticular tissue affected by a disease or disorder. In specificembodiments, the marker is a cell surface antigen that is differentiallyor preferentially expressed by specific cell types. For example, aleukemia cancer stem cell differentially expresses CD123 relative to anormal hematopoietic stem cell.

As used herein, the term “marker phenotype” in the context of a tissue(e.g., a normal or cancer cell or a tumor cell) means any combination ofantigens (e.g., receptors, ligands, and other cell surface markers),molecules, or other chemical or biological entities that arespecifically found in or on a tissue that it is desired to identify aparticular tissue affected by a disease or disorder. In specificembodiments, the marker phenotype is a cell surface phenotype. Inaccordance with this embodiment, the cell surface phenotype may bedetermined by detecting the expression of a combination of cell surfaceantigens. Non-limiting examples of cell surface phenotypes of cancerstem cells of certain tumor types include CD34⁺/CD38⁻, CD123+,CD44⁺/CD24⁻, CD133⁺, CD34⁺/CD10⁻/CD19⁻, CD138⁻/CD34⁻/CD19⁺, CD133⁺/RC2⁺,CD44⁺/α₂β₁ ^(hi)/CD133⁺, CLL-1, SLAMs, and other cancer stem cellsurface phenotypes mentioned herein, as well as those that are known inthe art.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopeia, or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofa cancer or a symptom thereof in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or a combination of therapies (e.g., a combination of prophylactic ortherapeutic agents). In some embodiments, such terms refer to one, two,three, or more results following the administration of one or moretherapies: (1) a stabilization, reduction or elimination of the cancerstem cell population, (2) a stabilization, reduction or elimination inthe cancer cell population, (3) an increase in response rate, (4) anincrease in the length or duration of remission, (5) a decrease in therecurrence rate of cancer, (6) an increase in the time to recurrence ofcancer, (7) an increase in the disease-free, relapse-free,progression-free, and/or overall survival of the patient, and (8) anamelioration of cancer-related symptoms and/or quality of life. Inspecific embodiments, such terms refer to a stabilization, reduction orelimination of the cancer stem cell population.

As used herein, the terms “fragment” and “portion” in the context ofproteinaceous agents refer to an amino acid sequence comprising an aminoacid sequence of at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 1 5 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino acid residues, at least 70 contiguous amino acidresidues, at least 80 contiguous amino acid residues, at least 90contiguous amino acid residues, at least 100 contiguous amino acidresidues, at least 125 contiguous amino acid residues, at least 150contiguous amino acid residues, at least 175 contiguous amino acidresidues, at least 200 contiguous amino acid residues, or at least 250contiguous amino acid residues of a protein or polypeptide.

As used herein, the term “predetermined reference range” refers to areference range for the particular biological entity, e.g., cancer stemcell, for a subject or a population of subjects. Each laboratory mayestablish its own reference range for each particular assay, or astandard reference range for each assay may be made available and usedlocally, regionally, nationally, or worldwide or may bepatient-specific. In one specific embodiment, the term refers to areference range for the amount of cancer stem cells in a patient (e.g.,as determined by in vivo imaging) or a specimen from a patient. Inanother specific embodiment, the term refers to a reference range forthe amount of cancer cells in a patient (e.g. as described by in vivoimaging) or a specimen from a patient.

As used herein, the term “prophylactically effective regimen” refers toan effective regimen for dosing, timing, frequency and duration of theadministration of one or more therapies for the prevention of cancer ora symptom thereof. In a specific embodiment, the regimen achieves one,two, three, or more of the following results: (1) a stabilization,reduction or elimination of the cancer stem cell population, (2) astabilization, reduction or elimination in the cancer cell population,(3) an increase in response rate, (4) an increase in the length orduration of remission, (5) a decrease in the recurrence rate of cancer,(6) an increase in the time to recurrence of cancer, (7) an increase inthe disease-free, relapse-free, progression-free, and/or overallsurvival of the patient, and (8) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the term “stabilizing” and analogous terms, when used inthe context of a cancer stem cell population or cancer cell population,refer to the prevention of an increase in the cancer stem cellpopulation or cancer cell population, respectively. In other words, theamount of cancer stem cells or the amount of cancer cells that a canceris composed of is maintained, and does not increase, or increases byless than 10%, preferably less than 5%.

As used herein, the term “therapeutically effective regimen” refers to aregimen for dosing, timing, frequency, and duration of theadministration of one or more therapies for the treatment and/ormanagement of cancer or a symptom thereof. In a specific embodiment, theregimen achieves one, two, three, or more of the following results: (1)a stabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate, (10) a decrease in hospitalization lengths, (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, and (12) a increase in the number of patientsin remission.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “subject” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a cancer or one or moresymptoms thereof. In certain embodiments, the terms “therapy” and“therapies” refer to chemotherapy, small molecule therapy,radioimmunotherapy, toxin therapy, prodrug-activating enzyme therapy,biologic therapy, antibody therapy, surgical therapy, hormone therapy,immunotherapy, anti-angiogenic therapy, targeted therapy, epigenetictherapy, demethylation therapy, histone deacetylase inhibitor therapy,differentiation therapy, radiation therapy, or a combination of theforegoing and/or other therapies useful in the prevention, managementand/or treatment of a cancer or one or more symptoms thereof.

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of cancer,the reduction or amelioration of the severity of cancer, and/or theamelioration of one or more symptoms thereof resulting from theadministration of one or more therapies. In specific embodiments, suchterms refer to one, two or three or more results following theadministration of one, two, three, or more therapies: (1) astabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate, (10) a decrease in hospitalization lengths, (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%, and (12) an increase in the number of patientsin remission. In certain embodiments, such terms refer to astabilization or reduction in the cancer stem cell population. In someembodiments, such terms refer to a stabilization or reduction in thegrowth of cancer cells. In some embodiments, such terms refer to astabilization or reduction in the cancer stem cell population and areduction in the cancer cell population. In some embodiments, such termsrefer to a stabilization or reduction in the growth and/or formation ofa tumor. In some embodiments, such terms refer to the eradication,removal, or control of primary, regional, or metastatic cancer (e.g.,the minimization or delay of the spread of cancer). In some embodiments,such terms refer to a reduction in mortality and/or an increase insurvival rate of a patient population. In further embodiments, suchterms refer to an increase in the response rate, the durability ofresponse, or number of patients who respond or are in remission. In someembodiments, such terms refer to a decrease in hospitalization rate of apatient population and/or a decrease in hospitalization length for apatient population.

Concentrations, amounts, cell counts, percentages and other numericalvalues may be presented herein in a range format. It is to be understoodthat such range format is used merely for convenience and brevity andshould be interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. A model of the three-dimensional structure of diphtheria toxin(DT) based on the X-ray crystallographic coordinates. Alpha-carbonbackbone is shown with flat arrows for beta-sheets and cylinders foralpha-helices. Catalytic, translocation and receptor-binding domains areshown.

FIG. 2. Mechanism of cell intoxication by DT. Steps include (a) cellbinding, (b) receptor-mediated endocytosis, (c) low pH, furin andthioredoxin reductase, beta-COP, and Hsp90 mediated translocation, (d)refolding and ADP ribosylation of EF2, and (e) cell death. (Ratts etal., “A conserved motif in transmembrane helix 1 of diphtheria toxinmediates catalytic domain delivery to the cytosol,” Proc. Natl. Acad.Sci. U.S.A. 102:15635-15640 (2005).

FIG. 3. A model of DT₃₈₈IL-3. Alpha-carbon backbone shown with blue forDT catalytic domain, green for DT translocation domain, and white forIL-3. Model based on X-ray coordinates for DT and human IL-3. (Choe etal., “The crystal structure of diphtheria toxin,” Nature 357:216-222(1992).

FIG. 4. A photomicrograph of a bone marrow biopsy from a patient, priorto treatment (A) and two months post-treatment (B). Wright-Giemsastained at 400× magnification.

FIG. 5. Graph showing percentage of patients with grade 2 or lowerdrug-related toxicities for DT₃₈₈IL-3 in connection with Example 2,infra.

FIG. 6A-B. Graph showing serum levels of DT₃₈₈IL-3 on Day 1 (A) and Day12 (B) in connection with Example 2, infra.

FIGS. 7A-C. Pharmacokinetics and immune response in connection withExample 2, infra. FIG. 7A: Cmax (μg/ml) as a function of dose (μg/kg).FIG. 7B: Cmax on Day 12 versus Cmaxon Day 1, showing relationshipbetween Cmaxon first and last dose. FIG. 7C: Peak Serum DT₃₈₈IL-3(μg/ml) versus pretreatment serum anti-diphtheria toxin (anti-DT)antibody (μg/ml), showing relationship between peak drug andpretreatment antibody levels.

FIG. 8A: A photomicrograph of pre- and post-marrow aspirate from patient#19 in connection with Example 2, infra. FIG. 8B: Blood counts forpatient #19 in connection with Example 2, infra. FIG. 8C: Aphotomicrograph of pre- and post-marrow aspirate from patient #36 inconnection with Example 2, infra. FIG. 8D: Blood counts for patient #36in connection with Example 2, infra.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for inhibitinginterleukin-3 receptor-expressing cells comprising administering to ahuman in need of such inhibition a pharmaceutical composition comprisingan amount of a human interleukin-3-diphtheria toxin conjugate effectivein inhibiting said cells and a pharmaceutically acceptable carrier,wherein the cells express the alpha subunit (in specific embodiments,the alpha and beta subunits) of the interleukin-3 receptor. Othermethods include treating, preventing and/or managing a disease ordisorder that displays or is characterized by interleukin-3receptor-expressing cells by administering to a human in need of suchtreatment, prevention and/or management a pharmaceutical compositioncomprising an amount of a human interleukin-3-diphtheria toxin conjugateeffective in inhibiting said cells and a pharmaceutically acceptablecarrier, wherein the cells express the alpha subunit (in specificembodiments, the alpha and beta subunits) of the interleukin-3 receptor.Such diseases and disorders include, but are not limited to, cancer,autoimmune disease, inflammatory disease, and allergic disease. Thepresent invention is also directed to methods for purging bone marrow orperipheral blood, by contacting ex vivo the bone marrow or peripheralblood sample obtained from a human with a composition comprising anamount of an interleukin-3-diphtheria toxin conjugate for a timesufficient to significantly purge the bone marrow or peripheral blood ofcells expressing the alpha subunit (in specific embodiments, the alphaand beta subunits) of the interleukin-3 receptor. Accordingly, thepresent invention is also directed to a method for performing anautologous bone marrow transplant by administering back into the patientsuch purged bone marrow or peripheral blood, as well as compositionscomprising such purged bone marrow or peripheral blood optionally with apharmaceutically acceptable carrier.

5.1 Interleukin-3-Diphtheria Toxin Conjugate

In one embodiment, an interleukin-3-diphtheria toxin conjugate of thepresent invention comprises the full-length, mature (lacking the signalpeptide) interleukin-3 protein (IL-3), or a portion, analog orderivative thereof that binds to the interleukin-3 receptor or a subunitthereof expressed on a cell surface, conjugated through a recombinanttechnology or through chemical (covalent) bond to diphtheria toxin, or aportion, analog or derivative thereof, which toxin preferably lacks thenative cell binding domain. In a preferred embodiment, IL-3 is humanIL-3. In certain embodiments, the conjugate comprises the catalytic andtranslocation domains of diphtheria toxin fused via a covalent bond tohuman IL-3. In other embodiments, the diphtheria toxin is linked via apeptide linker to the human IL-3 portion of the conjugate. The linkerfor the conjugate may be two, three, five, ten, or fifteen amino acidsin length. The length of the linker may vary to provide optimal bindingof the conjugate. In a preferred aspect, the peptide linker is two tofour amino acids long. In a more specific aspect, the peptide linker isa Met-His linker. Although not intending to be bound by a particularmechanism of action, the flexible peptide linker facilitates chainpairing and minimizes possible refolding. Linker molecules are commonlyknown in the art and described in Denardo et al., 1998, Clin. CancerRes. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; andZimmerman et al., 1999, Nucl. Med. Biol. 26:943-50 each incorporated byreference in their entireties.

In other embodiments, the invention provides pharmaceutical compositionscomprising a conjugate of the invention and a pharmaceuticallyacceptable carrier. In accordance with the present invention, theconjugate can comprise any domain of DT linked via any linker moleculeknown in the art to any domain of IL-3. In a specific embodiment, theconjugate is DT₃₈₈IL-3 (FIG. 3), which is a fusion protein comprisingamino acids 1-388 fused to full-length, mature, human IL-3 via a Met-Hisamino acid linker.

Diphtheria toxin (DT) is a 535 amino acid protein with three domainsconsisting of a catalytic domain (amino acids 1-186) connected by anarginine-rich disulfide loop to a translocation domain (amino acids187-388) followed by a cell binding domain (amino acids 389-535; FIG.1). See, e.g., Choe et al., “The crystal structure of diphtheria toxin,”Nature 357:216-222 (1992). The amino acid sequence of DT can be found inthe GenBank database (see, e.g., Accession No. AAN28949). Fragments,analogs and derivatives of diphtheria toxin can be useful in the presentapplication. In some embodiments, the conjugate of the inventionconsists of the catalytic, the translocation and the cell bindingdomains of DT. In other embodiments, the conjugate consists of the cellbinding and the catalytic domains of DT. In yet other embodiments, theconjugate of the invention consists of the cell binding and thetranslocation domains of DT. In preferred embodiments, the conjugate ofthe invention consists of the catalytic and translocation domains of DT.In some embodiments, the conjugate of the invention comprises one ofeither the translocation, catalytic, or cell binding domain.

Fragments, analogs, and derivatives of IL-3 can be useful in the presentinvention provided that when fused to the diphtheria toxin portion ofthe conjugate, such fragments, analogs and derivatives maintain theability to bind a subunit of the IL-3 receptor or the native IL-3receptor expressed on the surface of a cell. Preferably, the bindingkinetics of the fragments, analogs or derivatives remain the same orvary only by not more than 25%. The IL-3 polypeptide may be from anyspecies. The nucleotide and/or amino acid sequences of IL-3 polypeptidescan be found in the literature or public databases, or the nucleotideand/or amino acid sequences can be determined using cloning andsequencing techniques known to one of skill in the art. In someembodiments, the IL-3 is a mammalian IL-3. In a preferred embodiment, anIL-3 polypeptide is human IL-3, an analog, derivative, or a fragmentthereof. The amino acid sequence of human IL-3 can be found in theGenBank database (see, e.g., Accession No. AAC08706).

In one embodiment of the invention, an IL-3 polypeptide comprises anamino acid sequence which contains at least one conservative amino acidsubstitution, but not more than 50 conservative amino acidsubstitutions, even more preferably, not more than 40 conservative aminoacid substitutions, still more preferably, not more than 30 conservativeamino acid substitutions, and still even more preferably, not more than20 conservative amino acid substitutions relative to the native IL-3amino acid sequence (e.g., the native human IL-3 amino acid sequence),which result in a silent change, i.e., no change in activity. In anotherembodiment of the invention, an IL-3 polypeptide comprises an amino acidsequence which contains at least one conservative amino acidsubstitution; but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1conservative amino acid substitutions relative to the native IL-3 aminoacid sequence (e.g., the native human IL-3 amino acid sequence), whichresult in a silent change. In yet another embodiment, an IL-3polypeptide comprises an amino acid sequence which contains one or moreconservative substitutions or a combination of non-conservative andconservative amino acid substitutions relative to the native IL-3 aminoacid sequence, which results in a silent change.

To improve or alter the characteristics of IL-3 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins” including single or multiple amino acid substitutions,deletions, additions, or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity, potency, affinity, and/or increasedstability. In addition, these may be purified in higher yields and showbetter solubility than the corresponding natural polypeptide, forexample, under certain purification and storage conditions. Forinstance, for many proteins, it is known in the art that one or moreamino acids may be deleted from the N-terminus or C-terminus withoutsubstantial loss of biological function. An exemplary conjugatecomprising a modified IL-3 with amino acid substitution K116W in humanIL-3. Another exemplary conjugate comprises human IL-3 missing aminoacids 125-133. Both of these conjugates with mutant IL-3 sequencesexhibit enhanced binding to the IL-3 receptor and exhibit greatercytotoxicity against leukemia cells. (For non-limiting examples ofconjugates, see Liu et al. “Diphtheria toxin fused to variantinterleukin-3 provides enhanced binding to the interleukin-3 receptorand more potent leukemia cell cytotoxicity,” Exp. Hematol. 32:277-281(2004); Hogge et al. “Variant diphtheria toxin-interleukin-3 fusionproteins with increased receptor affinity have enhanced cytotoxicityagainst acute myeloid leukemia progenitors,” Clin. Cancer Res.12:1284-1291 (2006); Testa et al. “Diphtheria toxin fused to varianthuman interleukin-3 induces cytotoxicity of blasts from patients withacute myeloid leukemia according to the level of interleukin-3 receptorexpression,” Blood 106:2527-2529 (2005); and Klein et al. “Receptorbinding kinetics of human IL-3 variants with altered proliferativeactivity,” Biochem. Biophys. Res. Comm. 288:1244-1249 (2001)).

In another embodiment, an IL-3 polypeptide is at least 50%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% identical to a native IL-3 amino acid sequence(e.g., a native human IL-3 amino acid sequence).

5.1.1 Methods for Producing Interleukin-3-Diphtheria Toxin Conjugates

The conjugates of the present invention can be made by standardrecombinant DNA techniques or by protein synthetic techniques, e.g., byuse of a peptide synthesizer. For example, a nucleic acid moleculeencoding a conjugate of the invention can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, e.g., Current Protocols in MolecularBiology, Ausubel et al., eds., John Wiley & Sons, 1992).

The nucleotide sequences encoding a conjugate of the invention (IL-3 anddiphtheria toxin sequences) may be obtained from any informationavailable to those of skill in the art (i.e., from Genbank, theliterature, or by routine cloning). The nucleotide sequence coding for aconjugate can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. In some instances,the diphtheria toxin sequence can be truncated in order to remove aspecific domain, such as the targeting domain. The techniques formodifying or truncating DNA are well known to those of skill in the artof molecular biology. Also, the IL-3 and the diphtheria toxin sequencescan be ligated in such a way as to generate a DNA sequence that, whentranslating, creates a polypeptide that is a compound of the invention.In preferred examples, a linker sequence is introduced into therecombinant sequence that links the IL-3 sequence and the diphtheriatoxin sequence. A variety of host-vector systems may be utilized in thepresent invention to express the protein-coding sequence. These includebut are not limited to mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); microorganisms such as yeast (e.g. Pichia)containing yeast vectors; or bacteria (such as E. coli) transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elementsof vectors vary in their strengths and specificities. Depending on thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used. In a specificembodiment, the protein is expressed in E. coli. In another specificembodiment, the protein is expressed in Pichia.

The expression of a conjugate of the invention may be controlled by anypromoter or enhancer element known in the art. Promoters which may beused to control expression of a conjugate include, but are not limitedto, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42), the tetracycline (Tet)promoter (Gossen et al., 1995, Proc. Nat. Acad. Sci. U.S.A.89:5547-5551); prokaryotic expression vectors such as the β-lactamasepromoter (VIIIa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25; see also “Useful proteins from recombinantbacteria,” in Scientific American, 1980, 242:74-94); plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58);alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286); neuronal-specific enolase (NSE) which is active inneuronal cells (Morelli et al., 1999, Gen. Virol. 80:571-83);brain-derived neurotrophic factor (BDNF) gene control region which isactive in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res.Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter whichis active in astrocytes (Gomes et al., 1999, Braz. J. Med. Biol. Res.32(5):619-631; Morelli et al., 1999, Gen. Virol. 80:571-83) andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason et al., 1986, Science 234:1372-1378). In aspecific embodiment, the expression of a conjugate of the invention isregulated by a constitutive promoter. In another embodiment, theexpression is regulated by an inducible promoter. In another embodiment,the expression is regulated by a tissue-specific promoter.

In a specific embodiment, a vector is used that comprises a promoteroperably linked to a conjugate-encoding nucleic acid, one or moreorigins of replication and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the polypeptide or fusion protein coding sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing the antibody molecule in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).Specific initiation signals may also be required for efficienttranslation of inserted fusion protein coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. Furthermore,the initiation codon must be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:51-544).

Expression vectors containing inserts of a gene encoding a conjugate canbe identified by three general approaches: (a) nucleic acidhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of a gene encoding a conjugate in an expression vector can bedetected by nucleic acid hybridization using probes comprising sequencesthat are homologous to an inserted gene encoding the conjugate. In thesecond approach, the recombinant vector/host system can be identifiedand selected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of a nucleotide sequence encoding a conjugate inthe vector. For example, if the nucleotide sequence encoding theconjugate is inserted within the marker gene sequence of the vector,recombinants containing the gene encoding the conjugate insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe gene product (e.g., conjugate) expressed by the recombinant. Suchassays can be based, for example, on the physical or functionalproperties of the conjugate in in vitro assay systems, e.g., binding toan antibody or the IL-3 receptor.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion proteins or conjugatesmay be controlled. Furthermore, different host cells have characteristicand specific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include, but are not limited to, CHO, VERY, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, NS0, and in particular, neuronal celllines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ humanneuroblastomas (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57),SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704:450-460), Daoy human cerebellar medulloblastoma (He et al., 1992, CancerRes. 52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992,In Vitro Cell. Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma(Cancer Res., 1970, 30: 2110-2118), 1321N1 human astrocytoma (Proc.Natl. Acad. Sci. U.S.A. 1977, 74: 4816), MOG-G-CCM human astrocytoma(Br. J. Cancer 1984, 49: 269), U87MG human glioblastoma-astrocytoma(Acta Pathol. Microbiol. Scand. 1968, 74: 465-486), A172 humanglioblastoma (Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 ratglioma cells (Benda et al., 1968, Science 161: 370-371), Neuro-2a mouseneuroblastoma (Proc. Natl. Acad. Sci. U.S.A. 1970, 65: 129-136), NB41A3mouse neuroblastoma (Proc. Natl. Acad. Sci. U.S.A. 1962, 48: 1184-1190),SCP sheep choroid plexus (Bolin et al., 1994, J. Virol. Methods 48:211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985, J.Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro18: 952-960), and normal cell lines such as, for example, CTX TNA2 ratnormal cortex brain (Radany et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89: 6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

For long-term, high-yield production of recombinant conjugates, stableexpression is preferred. For example, cell lines which stably expressthe conjugate of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express a conjugateof the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A. 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Proc. Natl. Acad. Sci. U.S.A. 77:3567; O'Hare, et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. U.S.A.78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Once a conjugate of the invention has been produced by recombinantexpression or by chemical synthesis, it may be purified by any methodknown in the art for purification of a protein, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

5.2 Pharmaceutical Compositions and Routes of Administration

The present invention provides compositions comprising a diphtheriatoxin-interleukin-3 conjugate of the invention. In particular, theinvention provides a pharmaceutical composition comprising an effectiveamount of a conjugate of the invention and a pharmaceutically acceptablecarrier or vehicle. In a specific embodiment, a pharmaceuticalcomposition comprises an effective amount of a conjugate of theinvention and a pharmaceutically acceptable carrier or vehicle. Thepharmaceutical compositions are suitable for veterinary and/or humanadministration. The pharmaceutical compositions are also suitable for exvivo purging of a bone marrow or peripheral blood sample, e.g. as may bepracticed prior to re-introduction of the purged sample as an autologoustransplant back into the patient as may be practiced following high dosechemotherapy for cancer.

The pharmaceutical compositions of the present invention can be in anyform that allows for the composition to be administered to a subject,said subject preferably being an animal, including, but not limited to ahuman, mammal, or non-human animal, such as a cow, horse, sheep, pig,fowl, cat, dog, mouse, rat, rabbit, guinea pig, etc., and is morepreferably a mammal, and most preferably a human.

The compositions of the invention can be in the form of a solid, liquid,or gas (aerosol). Typical routes of administration may include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal,ocular, intradermal, intratumoral, intracerebral, intrathecal, andintranasal. Parenteral administration includes subcutaneous injections,intravenous, intramuscular, intraperitoneal, intrapleural, intrasternalinjection or infusion techniques. In a specific embodiment, thecompositions are administered parenterally. In a more specificembodiment, the compositions are administered intravenously.Pharmaceutical compositions of the invention can be formulated so as toallow a compound of the invention to be bioavailable upon administrationof the composition to a subject. Compositions can take the form of oneor more dosage units, where, for example, a tablet can be a singledosage unit, and a container of a compound of the invention in aerosolform can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of subject (e.g.,human), the overall health of the subject, the type of cancer thesubject is in need of treatment of, the use of the composition as partof a multi-drug regimen, the particular form of the compound of theinvention, the manner of administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle may be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous, so as to provide an aerosol composition useful in, e.g.,inhalatory administration.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a compound of the invention is administered. Such pharmaceuticalcarriers can be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The carriers can besaline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea, and the like. In addition, auxiliary, stabilizing,thickening, lubricating and coloring agents can be used. In oneembodiment, when administered to a subject, the compounds of theinvention and pharmaceutically acceptable carriers are sterile. Water isa preferred carrier when the compound of the invention is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The composition may be intended for oral administration, and if so, thecomposition is preferably in solid or liquid form, where semi-solid,semi-liquid, suspension, and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer, or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as ethyl cellulose,carboxymethylcellulose, microcrystalline cellulose, or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it can contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol, cyclodextrin, or afatty oil.

The pharmaceutical composition can be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion, or suspension. The liquid can beuseful for oral administration or for delivery by injection. Whenintended for oral administration, a composition can comprise one or moreof a sweetening agent, preservatives, dye/colorant, and flavor enhancer.In a composition for administration by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer, and isotonic agent can also be included.

The liquid compositions of the invention, whether they are solutions,suspensions, or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides whichcan serve as the solvent or suspending medium, polyethylene glycols,glycerin, cyclodextrin, propylene glycol, or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates, or phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin an ampoule, a disposable syringe, or a multiple-dose vial made ofglass, plastic or other material. Physiological saline is a preferredadjuvant. An injectable composition is preferably sterile.

The pharmaceutical compositions comprise an effective amount of aconjugate of the invention such that a suitable dosage will be obtained(see Section 5.3.1, infra, for suitable dosages). Typically, this amountis at least 0.01% of a conjugate of the invention by weight of thecomposition. When intended for oral administration, this amount can bevaried to be between 0.1% and 80% by weight of the composition.Preferred oral compositions can comprise from between 4% and 50% of thecompound of the invention by weight of the composition. Preferredcompositions of the present invention are prepared so that a parenteraldosage unit contains from between 0.01% and 2% by weight of the compoundof the invention.

The compositions of the invention can be administered by any convenientroute, for example, by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucosa, rectal,and intestinal mucosa, etc.). Administration can be systemic or local.Various delivery systems are known, e.g., microparticles, microcapsules,capsules, etc., and may be useful for administering a compound of theinvention. In certain embodiments, more than one compound of theinvention is administered to a subject. Methods of administration mayinclude, but are not limited to, oral administration and parenteraladministration; parenteral administration including, but not limited to,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous;intranasal, epidural, sublingual, intranasal, intracerebral,intraventricular, intrathecal, intravaginal, transdermal, rectally, byinhalation, or topically to the ears, nose, eyes, or skin. The preferredmode of administration is left to the discretion of the practitioner,and will depend, in-part, upon the site of the medical condition (suchas the site of cancer, a cancerous tumor, or a pre-cancerous condition).

In one embodiment, the compounds of the invention are administeredparenterally. In a specific embodiment, the compounds of the inventionare administered intravenously. In another embodiment, the compounds ofthe invention are administered by continuous infusion. In a particularembodiment, the compounds of the invention are administered by aninfusion that lasts for about 15 minutes, about 20 minutes, about 30minutes, about 45 minutes, about 1 hour, or about 2 hours.

In specific embodiments, it can be desirable to administer one or morecompounds of the invention locally to the area in need of treatment.This can be achieved, for example, and not by way of limitation, bylocal infusion during surgery; topical application, e.g., in conjunctionwith a wound dressing after surgery; by injection; by means of acatheter; by means of a suppository; or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. In one embodiment,administration can be by direct injection at the site (or former site)of a cancer, tumor, or precancerous tissue. In certain embodiments, itcan be desirable to introduce one or more compounds of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection. Intraventricular injectioncan be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. In certainembodiments, one or more compounds of the invention can be injectedintraperitoneally.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the compounds of the invention can be formulated asa suppository, with traditional binders and carriers such astriglycerides.

In yet another embodiment, the compounds of the invention can bedelivered in a controlled release system. In one embodiment, a pump canbe used (see Sefton, CRC Crit. Ref Biomed. Eng. 1987, 14, 201; Buchwaldet al., Surgery 1980, 88: 507; Saudek et al., N. Engl. J. Med. 1989,321: 574). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York,1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 1983,23, 61; see also Levy et al., Science 1985, 228, 190; During et al.,Ann. Neurol. 1989, 25, 351; Howard et al., J. Neurosurg., 1989, 71,105). In yet another embodiment, a controlled-release system can beplaced in proximity of the target of the compounds of the invention,e.g., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, 1984, pp. 115-138). Other controlled-release systemsdiscussed in the review by Langer (Science 1990, 249, 1527-1533) canalso be used.

In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the compounds of the invention (see,e.g., U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No.5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCTPublication No. WO 99/15154; and PCT Publication No. WO 99/20253.Examples of polymers used in sustained release formulations include, butare not limited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In a preferred embodiment, the polymer usedin a sustained release formulation is inert, free of leachableimpurities, stable on storage, sterile, and biodegradable.

In a specific embodiment, a pump can be used to deliver the compounds ofthe invention (see, e.g, Sefton, CRC Crit. Ref Biomed. Eng. 1987, 14,201; Buchwald et al., Surgery 1980, 88: 507; Saudek et al., N. Engl. J.Med. 1989, 321: 574). In a specific embodiment, the pump may be, but isnot limited to, an insulin-like pump.

The present compositions can take the form of solutions, suspensions,tablets, pills, pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. In oneembodiment, the pharmaceutically acceptable carrier is a capsule (seee.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences by E. W. Martin.

Sustained or directed release compositions that can be formulatedinclude, but are not limited to, compounds of the invention protectedwith differentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc. It is also possible to freeze-dry thecompositions and use the lyophilizates obtained, for example, for thepreparation of products for injection.

In a preferred embodiment, the conjugates of the invention areformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to animals,particularly human beings. Typically, the carriers or vehicles forintravenous administration are sterile isotonic aqueous buffersolutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anaesthetic such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachet indicating the quantity ofactive agent. Where a conjugate of the invention is to be administeredby infusion, it can be dispensed, for example, with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where theconjugate of the invention is administered by injection, an ampoule ofsterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optional agents, for example, sweetening agents suchas fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving complex are also suitable for orally administered compositionsof the invention. In these later platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving complex, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be used. Oral compositions can includestandard carriers such as mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Such carriersare preferably of pharmaceutical grade.

The pharmaceutical compositions of the invention can be intended fortopical administration, in which case the carrier can be in the form ofa solution, emulsion, ointment, or gel base. The base, for example, cancomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents can be present in acomposition for topical administration. If intended for transdermaladministration, the composition can be in the form of a transdermalpatch or an iontophoresis device. Topical formulations can comprise aconcentration of a compound of the invention of from between 0.01% and10% w/v (weight per unit volume of composition).

The compositions can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.Aerosols of the compositions can be delivered in single phase,bi-phasic, or tri-phasic systems in order to deliver the composition.Delivery of the aerosol includes the necessary container, activators,valves, subcontainers, spacers and the like, which together can form akit. Preferred aerosols can be determined by one skilled in the art,without undue experimentation.

Whether in solid, liquid, or gaseous form, the compositions of thepresent invention can comprise an additional active agent selected fromamong those including, but not limited to, an additional prophylacticagent, an additional therapeutic agent, an antiemetic agent, ahematopoietic colony stimulating factor, an adjuvant therapy, a vaccineor other immune stimulating agent, an antibody/antibody fragment-basedagent, an anti-depressant and an analgesic agent. For instance in aparticular embodiment, the pharmaceutical composition comprises acompound of the invention, an additional agent, and a pharmaceuticallyacceptable acceptable carrier or vehicle.

The pharmaceutical compositions can be prepared using methodology wellknown in the pharmaceutical art. For example, a composition intended tobe administered by injection can be prepared by combining a compound ofthe invention with water so as to form a solution. A surfactant can beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are complexes that can non-covalently interactwith a compound of the invention so as to facilitate dissolution orhomogeneous suspension of the compound of the invention in the aqueousdelivery system.

In one embodiment, the pharmaceutical compositions of the presentinvention may comprise one or more known therapeutically active agents.

5.3 Therapeutic and Prophylatic Uses of Interleukin-3-Diphtheria ToxinFusion Conjugates

The present invention provides methods for inhibiting IL-3 receptorexpressing cells in a human in need thereof by administering aneffective amount of a human IL-3-diphtheria toxin conjugate of theinvention. In certain embodiments, the IL-3 receptor expressing cellsare not myeloid leukemia cells. In some embodiments, the cells expressthe alpha subunit of the interleukin-3 receptor. In other embodiments,the cells express the beta subunit of the interleukin-3 receptor. In yetother embodiments, the cells express both the alpha and beta subunits ofthe IL-3 receptor.

The present invention is directed to therapies which involveadministering one of more the interleukin-3-diphtheria toxin conjugatesof the invention and compositions comprising theinterleukin-3-diphtheria toxin conjugates to a subject, preferably ahuman subject, for preventing, treating, managing, and/or amelioratingdisease or disorder that displays or is characterized by interleukin-3receptor expression or one or more symptoms thereof. In one embodiment,the invention provides a method of preventing, treating, managing,and/or ameliorating a disease or disorder that displays or ischaracterized by interleukin-3 receptor expression or one or moresymptoms thereof, said method comprising administering to a subject inneed thereof an effective amount of one or more theinterleukin-3-diphtheria toxin conjugates of the invention. Suchdiseases and disorders include cancer, allergic diseases, inflammatorydiseases, and autoimmune diseases.

The invention also provides methods comprising administering to asubject in need thereof an interleukin-3-diphtheria toxin conjugate ofthe invention and one or more therapies (e.g., one or more prophylacticor therapeutic agents) other than the interleukin-3-diphtheria toxinconjugate of the invention that are currently being used, have beenused, are known to be useful, or may be useful in the prevention,treatment, management, and/or amelioration of a disease or disorder thatdisplays or is characterized by interleukin-3 receptor expression or oneor more symptoms thereof. The prophylactic or therapeutic agents of thecombination therapies of the invention can be administered sequentiallyor concurrently. In a specific embodiment, the combination therapies ofthe invention comprise an effective amount of a conjugate of theinvention and an effective amount of at least one other therapy whichhas the same mechanism of action as said conjugate. In a specificembodiment, the combination therapies of the invention comprise aneffective amount of a conjugate of the invention and an effective amountof at least one other therapy (e.g., prophylactic or therapeutic agent)which has a different mechanism of action than said conjugate. Incertain embodiments, the combination therapies of the present inventionimprove the prophylactic or therapeutic effect of a conjugate of theinvention by functioning together with the conjugate to have an additiveor synergistic effect. In certain embodiments, the combination therapiesof the present invention reduce the side effects associated with theprophylactic or therapeutic agents. In other embodiments, thecombination therapies are administered prior to, during, or after theadministration of the compositions of the invention.

Cancer or a neoplastic disease, including, but not limited to,neoplasms, tumors, metastases, or any disease or disorder characterizedby uncontrolled cell growth, can be treated, suppressed, delayed,managed, inhibited or prevented by administering to a subject in needthereof a prophylactically effective regimen or a therapeuticallyeffective regimen, the regimen comprising administering to the patient acompound of the invention. In specific embodiments, the inventionencompasses the treatment, suppression, delaying, management, inhibitingof growth and/or progression, and prevention of cancer or neoplasticdisease as described herein.

In one embodiment, the conjugates of the invention are administered asmonotherapy for the prevention, treatment, and/or management of cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention or apharmaceutical composition of the invention, wherein the patient hasbeen diagnosed with cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention or apharmaceutical composition of the invention, wherein the patient hasrelapsed from cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention or apharmaceutical composition of the invention, wherein the patient hasfailed or is failing therapy.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention or apharmaceutical composition of the invention, wherein the patient is inremission from cancer.

One aspect of the invention relates to a method of preventing, treating,and/or managing cancer in a patient (e.g., a human patient), the methodcomprising administering to the patient a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention or apharmaceutical composition of the invention, wherein the patient isrefractory to therapy.

In one embodiment, the cancer is a hematologic cancer. For instance, thecancer can be leukemia, lymphoma, myelodysplastic syndrome (MDS), ormyeloma. In another embodiment, the cancer is a solid tumor.

In one embodiment of this aspect, the patient has received or isreceiving another therapy. In another embodiment of this aspect, thepatient has not previously received a therapy for the prevention,treatment, and/or management of the cancer.

The medical practitioner can diagnose the patient using any of theconventional cancer screening methods including, but not limited tophysical examination (e.g., prostate examination, rectal examination,breast examination, lymph nodes examination, abdominal examination, skinsurveillance, testicular exam, general palpation), visual methods (e.g.,colonoscopy, bronchoscopy, endoscopy), PAP smear analyses (cervicalcancer), stool guaiac analyses, blood tests (e.g., complete blood count(CBC) test, prostate specific antigen (PSA) test, carcinoembryonicantigen (CEA) test, cancer antigen (CA)-125 test, alpha-fetoprotein(AFP), liver function tests), karyotyping analyses, bone marrow analyses(e.g., in cases of hematological malignancies), histology, cytology,flow cytometry, a sputum analysis, and imaging methods (e.g., computedtomography (CT), magnetic resonance imaging (MRI), ultrasound, X-rayimaging, mammography, PET scans, bone scans, radionuclide scans).

Another aspect of the invention relates to a method of preventing,treating, and/or managing a solid tumor in a patient (e.g., a humanpatient), the method comprising administering to a patient in needthereof a prophylactically effective regimen or a therapeuticallyeffective regimen, the regimen comprising administering to the patient aconjugate or pharmaceutical composition of the invention wherein thepatient has been diagnosed with a solid tumor, and wherein the patienthas undergone a primary therapy to reduce the bulk of the tumor. Theprimary therapy to reduce the tumor bulk size is preferably a therapyother than a conjugate of the invention. In specific embodiment of thisaspect, the solid tumor is fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, or retinoblastoma.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer, the method comprising administering toa patient in need thereof a prophylactically effective regimen or atherapeutically effective regimen, the regimen comprising administeringto the patient a conjugate of the invention, wherein the patientreceived another therapy. In some embodiments, the prior therapy is, forexample, chemotherapy, small molecule therapy, radioimmunotherapy, toxintherapy, prodrug-activating enzyme therapy, biologic therapy, antibodytherapy, surgical therapy, hormone therapy, immunotherapy,anti-angiogenic therapy, targeted therapy, epigenetic therapy,demethylation therapy, histone deacetylase inhibitor therapy,differentiation therapy, radiation therapy, or any combination thereof.

In some embodiments, the prior therapy has failed in the patient. Insome embodiments, the therapeutically effective regimen comprisingadministration of a conjugate of the invention is administered to thepatient immediately after the patient has undergone the prior therapy.For instance, in certain embodiments, the outcome of the prior therapymay be unknown before the patient is administered the conjugate.

Another aspect of the invention relates to a method of preventing cancerin a patient (e.g., a human patient), the method comprisingadministering to a patient in need thereof a prophylactically effectiveregimen or a therapeutically effective regimen, the regimen comprisingadministering to the patient a conjugate of the invention, wherein thecancer in the patient has entered remission. In some embodiments of thisaspect, through administration of a prophylactically effective regimenor a therapeutically effective regimen, the medical practitioner caneffectively cure the cancer, or prevent its reoccurrence.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer in a patient (e.g., a human patient),the method comprising administering to a patient in need thereof aprophylactically effective regimen or a therapeutically effectiveregimen, the regimen comprising administering to the patient a compoundor composition of the invention, wherein the conjugate is administeredat a dose that is lower than the maximum tolerated dose (MTD) over aperiod of three months, four months, six months, nine months, 1 year, 2years, 3 years, 4 years, or more.

Another aspect of the invention relates to a method of preventing,treating, and/or managing cancer in a patient (e.g., a human patient),the method comprising administering to a patient in need thereof aprophylactically effective regimen or a therapeutically effectiveregimen, the regimen comprising administering to the patient a conjugateof the invention, wherein the conjugate is administered at a dose thatis lower than the human equivalent dosage (HED) of the no observedadverse effect level (NOAEL) over a period of three months, four months,six months, nine months, 1 year, 2 years, 3 years, 4 years, or more. TheNOAEL, as determined in animal studies, is useful in determining themaximum recommended starting dose for human clinical trials. Forinstance, the NOAELs can be extrapolated to determine human equivalentdosages. Typically, such extrapolations between species are conductedbased on the doses that are normalized to body surface area (i.e.,mg/m²). In specific embodiments, the NOAELs are determined in mice,hamsters, rats, ferrets, guinea pigs, rabbits, dogs, primates (monkeys,marmosets, squirrel monkeys, baboons), micropigs, or minipigs. For adiscussion on the use of NOAELs and their extrapolation to determinehuman equivalent doses, see Guidance for Industry Estimating the MaximumSafe Starting Dose in Initial Clinical Trials for Therapeutics in AdultHealthy Volunteers, U.S. Department of Health and Human Services Foodand Drug Administration Center for Drug Evaluation and Research (CDER),Pharmacology and Toxicology, July 2005.

While not being bound by any specific theory, Applicants believe that bythe administration of the prophylactically and/or therapeuticallyeffective regimens, the cancer stem cell population of a cancer/tumor isstabilized or reduced, so as to limit or prevent the potentialrepopulation of the tumor.

In certain embodiments of these aspects, the regimens compriseadministering a prophylactically effective regimen and/or atherapeutically effective regimen, wherein the regimen results in areduction in the cancer stem cell population in the patient. In oneembodiment, the patient undergoing the regimen is monitored to determinewhether the regimen has resulted in a reduction in the cancer stem cellpopulation in the patient.

Typically, the monitoring of the amount of cancer stem cells isconducted by detecting the amount of cancer stem cells in a specimenextracted from the patient. Methods of detecting the amount of cancerstem cells in a specimen are described infra in Section 5.4. Thismonitoring step is typically performed at least 1, 2, 4, 6, 7, 8, 10,12, 14, 15, 16, 18, 20, or 30, 60, 90, 120 days, 6 months, 9 months, 12months, or >12 months after the patient begins receiving the regimen.

In some embodiments, the specimen may be a blood specimen, wherein theamount of cancer stem cells per unit of volume (e.g., 1 ml) or othermeasured unit (e.g., per unit field in the case of a histologicalanalysis) is quantitated. In certain embodiments, the amount of cancerstem cells is determined as a portion (e.g., a percentage) of the cancercells present in the blood specimen, as a subset of the cancer cellspresent in the blood specimen, or as a subset of a subset of the cancercells present in the blood specimen. The amount of cancer stem cells, inother embodiments, can be determined as a percentage of the total bloodcells.

In other embodiments, the specimen extracted from the patient is atissue specimen (e.g., a biopsy extracted from suspected canceroustissue), where the amount of cancer stem cells can be measured, forexample, on the basis of the amount of cancer stem cells per unit weightof the tissue. In certain embodiments, the amount of cancer stem cellsis determined as a portion (e.g., a percentage) of the cancer cellspresent in the tissue, as a subset of the cancer cells present in thetissue, or as a subset of a subset of the cancer cells present in thetissue.

The amount of cancer stem cells in the extracted specimen can becompared with the amount of cancer stem cells measured in referencesamples to assess the efficacy of the regimen, and the amelioration ofthe cancer under therapy. In one embodiment, the reference sample is aspecimen extracted from the patient undergoing therapy, wherein thespecimen is extracted from the patient at an earlier time point (e.g.,prior to receiving the regimen, as a baseline reference sample, or at anearlier time point while receiving the therapy). In another embodiment,the reference sample is extracted from a healthy, noncancer-afflictedpatient.

In other embodiments the amount of cancer stem cells in the extractedspecimen can be compared with a predetermined reference range. In aspecific embodiment, the predetermined reference range is based on i)the amount of cancer stem cells obtained from a population(s) ofpatients suffering from the same type of cancer as the patientundergoing the therapy, or ii) the amount of stem cells obtained from apopulation(s) of patients without cancer.

If the reduction in the amount of cancer stem cells is determined to betoo small upon comparing the amount of cancer stem cells in the specimenextracted from the patient undergoing the regimen with the referencespecimen, then the medical practitioner has a number of options toadjust the regimen. For instance, the medical practitioner can thenincrease either the dosage of the compound or composition of theinvention administered, the frequency of the administration, theduration of administration, or any combination thereof. In a specificembodiment, after the determination is made, a second effective amountof a compound or composition of the invention can be administered to thepatient.

In certain embodiments, if the reduction in the amount of cancer stemcells is determined to be acceptable upon comparing the amount of cancerstem cells in the sample obtained from the patient undergoing thetherapeutic or prophylactic regimen with the reference sample, then themedical practitioner may elect not to adjust the regimen. For instance,the medical practitioner may elect not to increase either the dosage ofthe compound or composition of the invention being administered, thefrequency of the administration, the duration of administration, or anycombination thereof. Further, the medical practitioner may elect to addadditional therapies or combine therapies.

In other embodiments, the regimens comprise administering aprophylactically effective regimen and/or a therapeutically effectiveregimen, wherein the regimen results in a reduction in the amount ofcancer cells in the patient. In one embodiment, the patient undergoingthe regimen is monitored to determine whether the regimen has resultedin a reduction in the amount of cancer cells in the patient.

Typically, the monitoring of the amount of cancer cells is conducted bydetecting the amount of cancer cells in a specimen extracted from thepatient. Methods of detecting the amount of cancer cells in a specimenare described infra in Section 5.5. This monitoring step is typicallyperformed at least 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 16, 18, 20, or 30,60, 90, 120 days, 6 months, 9 months, 12 months, or >12 months after thepatient begins receiving the regimen.

In some embodiments, the specimen may be a blood specimen, wherein theamount of cancer cells per unit of volume (e.g., 1 ml) or other measuredunit (e.g., per unit field in the case of a histological analysis) isquantitated. The cancer cell population, in certain embodiments, can bedetermined as a percentage of the total blood cells.

In some embodiments, the sample obtained from the patient may be a bonemarrow specimen, wherein the amount of cancer cells per unit of volume(e.g., 1 ml) or other measured unit (e.g., per unit field in the case ofa histological analysis) is quantitated. The cancer cell population, incertain embodiments, can be determined as a percentage of the total bonemarrow cells.

In other embodiments, the specimen extracted from the patient is atissue specimen (e.g., a biopsy extracted from suspected canceroustissue), where the amount of cancer cells can be measured, for example,on the basis of the amount of cancer cells per unit weight of thetissue. The amount of cancer cells can also be measured usingimmunohistochemistry or flow cytometry.

The amount of cancer cells in the extracted specimen can be comparedwith the amount of cancer cells measured in reference samples to assessthe efficacy of the regimen and amelioration of the cancer undertherapy. In one embodiment, the reference sample is a specimen extractedfrom the patient undergoing therapy, wherein the specimen from thepatient is extracted at an earlier time point (e.g., prior to receivingthe regimen, as a baseline reference sample, or at an earlier time pointwhile receiving the therapy). In another embodiment, the referencesample is extracted from a healthy, noncancer-afflicted patient.

In other embodiments the cancer cell population in the extractedspecimen can be compared with a predetermined reference range. In aspecific embodiment, the predetermined reference range is based on theamount of cancer cells obtained from a population(s) of patientssuffering from the same type of cancer as the patient undergoing thetherapy.

If the reduction in the cancer cell population is judged too small uponcomparing the amount of cancer cells in the specimen extracted from thepatients undergoing therapy with the reference specimen, then themedical practitioner has a number of options to adjust the therapeuticregimen. For instance, the medical practitioner can then either increasethe dosage of the compound or composition of the invention administered,the frequency of the administration, the duration of administration, orany combination thereof. In a specific embodiment, after thedetermination is made, a second effective amount of a compound orcomposition of the invention can be administered to the patient.

If the reduction in the cancer cell population is judged to be adequateupon comparing the amount of cancer cells in the specimen extracted fromthe patients undergoing therapy with the reference specimen, then themedical practitioner may elect not to adjust the therapeutic regimen.For instance, the medical practitioner may elect not to increase thedosage of the compound or composition of the invention administered, thefrequency of the administration, the duration of administration, or anycombination thereof.

The above monitoring methods can also be used to monitor the amount ofinterleukin-3 receptor-expressing cells where the disease or disorder isnot a cancer, i.e., in allergic disease or autoimmune disease.

In embodiments, the medical practitioner may elect to measure the cancerpopulation using in vivo imaging techniques. For example, a ligand for atumor marker can be conjugated to a radioisotope, photon emittingcompound, or other signal emitting compound, and then the ligand can beinjected into the patient. The cancer cells can then be quantitated bymeasuring the signal generated when the ligand binds to the cancer cellsin vivo.

5.3.1 Dosage and Frequency of Administration

The amount of a diphtheria toxin-interleukin-3 pharmaceuticalcomposition of the invention used in the prophylactic and/or therapeuticregimens which will be effective in the prevention, treatment, and/ormanagement of diseases or disorders characterized by cells expressingthe interleukin-3 receptor beta subunit, including cancer, can bedetermined by methods disclosed herein. The frequency and dosage willvary according to factors specific for each patient depending on thespecific conjugates administered, the severity of the (e.g. cancerous)condition, the route of administration, as well as age, body, weight,response, and the past medical history of the patient. For example, thedosage of a conjugate of the invention which will be effective in thetreatment, prevention, and/or management of cancer can be determined byadministering the compound in an animal model such as, e.g., the animalmodels disclosed herein or known in to those skilled in the art. SeeSection 5.7.2, infra. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. See Section 5.7.1,infra.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise titrating the dosages administered to the patient so as toachieve a specified measure of therapeutic efficacy. Such measuresinclude a reduction in the amount of cancer stem cells in or from thepatient and/or a reduction in the amount of cancer cells in or from thepatient.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise administering dosages and regimens of a conjugate orpharmaceutical composition of the invention that are effective to reducecancer stem cels. Methods that can be used to determine the amount ofcancer stem cells in a patient prior to, during, and/or followingtherapy are discussed infra in Section 5.4.

In certain embodiments, the dosage of the conjugate of the invention inthe prophylactic and/or therapeutic regimen is adjusted so as to achievea reduction in the amount of cancer stem cells found in a test specimenextracted from a patient after undergoing the therapeutic regimen, ascompared with a reference sample. Here, the reference sample is aspecimen extracted from the patient undergoing therapy, wherein thespecimen is extracted from the patient at an earlier time point. In oneembodiment, the reference sample is a specimen extracted from the samepatient, prior to receiving the prophylactic or therapeutic regimen. Inspecific embodiments, the amount of cancer stem cells in the testspecimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% lower than in the reference sample.

In other embodiments, the dosage of the conjugate of the invention inthe prophylactic and/or therapeutic regimen is adjusted so as to achievea reduction in the amount of cancer stem cells found in a test specimenextracted from a patient after undergoing the prophylactic and/ortherapeutic regimen, as compared with a reference sample, wherein thereference sample specimen is extracted from a healthy,noncancer-afflicted patient. In specific embodiments, the amount ofcancer stem cells in the test specimen is at least within 60%, 50%, 40%,30%, 20%, 15%, 10%, 5%, or 2% of the amount of cancer stem cells in thereference sample.

In some embodiments, the dosage of the conjugate of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve anamount of cancer stem cells that falls within a predetermined referencerange. In these embodiments, the amount of cancer stem cells in a testspecimen is compared with a predetermined reference range. In a specificembodiment, the predetermined reference range is based on the amount ofcancer stem cells obtained from a population(s) of patients sufferingfrom the same type of cancer as the patient undergoing the therapy.

In some embodiments, the prophylactic and/or therapeutic regimenscomprise administering dosages of a conjugate or pharmaceuticalcomposition of the invention that are effective to reduce the cancercell population. Methods that can be used to determine the cancer cellpopulation in a patient undergoing treatment are discussed infra inSection 5.5.

In certain embodiments, the dosage of the conjugate of the invention inthe prophylactic and/or therapeutic regimen is adjusted so as to achievea reduction in the amount of cancer cells found in a test specimenextracted from a patient after undergoing the prophylactic and/ortherapeutic regimen, as compared with a reference sample. Here, thereference sample is a specimen extracted from the patient undergoingtherapy, wherein the specimen is extracted from the patient at anearlier time point. In one embodiment, the reference sample is aspecimen extracted from the same patient, prior to receiving theprophylactic and/or therapeutic regimen. In specific embodiments, theamount of cancer cells in the test specimen is at least 2%, 5%, 10%,15%, 20%, 30%, 40%, 50%, or 60% lower than in the reference sample.

In some embodiments, the dosage of the conjugate of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve anamount of cancer cells that falls within a predetermined referencerange. In these embodiments, the amount of cancer cells in a testspecimen is compared with a predetermined reference range.

In other embodiments, the dosage of the conjugate of the invention inprophylactic and/or therapeutic regimen is adjusted so as to achieve areduction in the amount of cancer cells found in a test specimenextracted from a patient after undergoing the prophylactic and/ortherapeutic regimen, as compared with a reference sample, wherein thereference sample is a specimen extracted from a healthy,noncancer-afflicted patient. In specific embodiments, the amount ofcancer cells in the test specimen is at least within 60%, 50%, 40%, 30%,20%, 15%, 10%, 5%, or 2% of the amount of cancer cells in the referencesample.

In treating certain human patients having solid tumors, extractingmultiple tissue specimens from a suspected tumor site may or may notprove impracticable. In these embodiments, the dosage of the compoundsof the invention in the prophylactic and/or therapeutic regimen for ahuman patient is extrapolated from doses in animal models that areeffective to reduce the amount of cancer stem cells in those animalmodels. In the animal models, the prophylactic and/or therapeuticregimens are adjusted so as to achieve a reduction in the amount ofcancer stem cells found in a test specimen extracted from an animalafter undergoing the prophylactic and/or therapeutic regimen, ascompared with a reference sample. The reference sample can be a specimenextracted from the same animal, prior to receiving the prophylacticand/or therapeutic regimen. In specific embodiments, the amount ofcancer stem cells in the test specimen is at least 2%, 5%, 10%, 15%,20%, 30%, 40%, 50%, or 60% lower than in the reference sample. The doseseffective in reducing the amount of cancer stem cells in the animals canbe normalized to body surface are (mg/m²) to provide an equivalent humandose.

The prophylactic and/or therapeutic regimens disclosed herein compriseadministration of a conjugate of the invention or pharmaceuticalcompositions thereof to the patient in a single dose or in multipledoses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses).

In one embodiment, the prophylactic and/or therapeutic regimens compriseadministration of a conjugate of the invention or pharmaceuticalcompositions thereof in multiple doses. When administered in multipledoses, the conjugate or pharmaceutical compositions are administeredwith a frequency and in an amount sufficient to prevent, treat, and/ormanage the condition. In one embodiment, the frequency of administrationranges from once a day up to about once every eight weeks. In anotherembodiment, the frequency of administration ranges from about once aweek up to about once every six weeks. In another embodiment, thefrequency of administration ranges from about once every three weeks upto about once every four weeks. In certain embodiments, the conjugate isadministered over a period of one week to two years. In yet anotherembodiment, the conjugate is administered over a period of two weeks orgreater. In other embodiments, the conjugate is administered over aperiod of two weeks to one year. In further embodiments, the conjugateis administered over a period of two weeks to six months. In someembodiments, the conjugate is administered over a period of two weeks totwelve weeks. In yet other embodiments, the conjugate is administeredover a period of two weeks to six weeks. In certain embodiments, theconjugate is administered once a week, twice a week, three times a week,four times a week, five times a week, six times a week, or seven times aweek. In preferred embodiments, the conjugate is administered at leastthree times a week. In other preferred embodiments, the compound isadministered daily for five consecutive days, or daily for sevenconsecutive days. In other embodiments, the conjugate is administeredonce a day, twice a day, three times a day, four times a day, or fivetimes a day. In preferred embodiments, the conjugate is administeredthree times a week over a period of two weeks. In some embodiments, eachtime the conjugate is administered, it is administered at a dose of 4μg/kg per day or greater. In some embodiments, the compound isadministered for one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or fifteen cycles.

In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. In a specific aspect, theconjugate can be administered at or below a dose that is the maximumdose tolerated without undue toxicity. In specific embodiments, wherethe disease or disorder is myeloid leukemia, the dosage given is in arange of between greater than 4 μg/kg per day to about 20 μg/kg per day.The per day dosages described herein may be administered on consecutiveand/or non-consecutive days. In a specific embodiment, a per day dosageis administered on non-consecutive days throughout a week, e.g., Monday,Wednesday, and Friday. In another specific embodiment, a per day dosageis administered on consecutive days throughout a week, e.g. Monday,Tuesday, Wednesday, Thursday, and Friday.

In specific aspects of this embodiment, the conjugate can beadministered at a dose of 4 μg/kg per day or greater. In other aspects,the conjugate can be administered at a dose in a range of about 4 μg/kgper day to about 20 μg/kg per day. In yet other aspects, the conjugatecan be administered at a dose in a range of about 4 μg/kg per day toabout 9 μg/kg per day. In yet other aspects, the conjugate can beadministered at a dose in a range of about 4 μg/kg per day to about 12.5μg/kg per day. In a specific aspect of this embodiment, the conjugatecan be administered at a dose of about 5.3 μg/kg per day, or at a doseof about 7.1 μg/kg per day, or at a dose of about 9.4 μg/kg per day, orat a dose of about 12.5 μg/kg per day. In a specific aspect, theconjugate can be administered at or below a dose that is the maximumdose tolerated without undue toxicity. In specific embodiments, wherethe disease or disorder is myeloid leukemia, the dosage given is in arange of between greater than 4 μg/kg to about 20 μg/kg.

In another embodiment, where the disease is myelodysplastic syndrome,the dosage given is at least 4 μg/kg or greater.

In some embodiments of the invention, the dosage of a conjugate of theinvention or pharmaceutical composition thereof administered is at least1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80,90 or 100 times lower than the maximum tolerated dose (MTD) over aperiod of one week, two weeks, one month, three months, four months, sixmonths, nine months, 1 year, 2 years, 3 years, 4 years, or more.

In some embodiments of the invention, the dosage of a conjugate of theinvention or pharmaceutical composition thereof administered is at least1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.8, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 20, 30,40, 50, 60, 70, 80, 90, or 100 times lower than the human equivalentdose (HED) of the no observed adverse effect level (NOAEL) over a periodof one week, two weeks, one month, three months, four months, sixmonths, nine months, 1 year, 2 years, 3 years, 4 years, or more. See thediscussion in Section 5.3, supra.

In certain embodiments, the dosage of a conjugate of the invention isadministered as an intravenous infusion over about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 120,180, or 240 minutes.

Generally, the dosage of a conjugate of the invention administered to asubject to prevent, treat, and/or manage cancer is in the range of 0.01to 500 μg/kg, and more typically, in the range of 0.1 μg/kg to 100μg/kg, of the subject's body weight. In one embodiment, the dosageadministered to a subject is in the range of 0.1 μg/kg to 50 μg/kg, or 1μg/kg to 50 μg/kg, of the subject's body weight, more preferably in therange of 0.1 μg/kg to 25 μg/kg, 1 μg/kg to 25 μg/kg, or 4 to 12.5 μg/kg,of the patient's body weight. In a preferred embodiment, the dosage ofconjugate of the invention administered to a subject is 4 μg/kg, 5.32μg/kg, 7.07 μg/kg, 9.4 μg/kg, or 12.5 μg/kg. of the patient's bodyweight.

In a specific embodiment, the dosage of a conjugate of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is 500 μg/kg or less, preferably 250 μg/kg or less, 100 μg/kg orless, 95 μg/kg or less, 90 μg/kg or less, 85 μg/kg or less, 80 μg/kg orless, 75 μg/kg or less, 70 μg/kg or less, 65 μg/kg or less, 60 μg/kg orless, 55 μg/kg or less, 50 μg/kg or less, 45 μg/kg or less, 40 μg/kg orless, 35 μg/kg or less, 30 μg/kg or less, 25 μg/kg or less, 20 μg/kg orless, 15 μg/kg or less, 12.5 μg/kg or less, 10 μg/kg or less, 9.4 μg/kgor less, 7.07 μg/kg or less, 5.32 μg/kg or less, 5 μg/kg or less, 4μg/kg or less, 2.5 μg/kg or less, 2 μg/kg or less, 1.5 μg/kg or less, or1 μg/kg or less, of a patient's body weight.

In a preferred embodiment, the dosage of conjugate of the inventionadministered to a subject to treat, prevent and/or manage cancer in apatient is a dose of 4 μg/kg, 5.32 μg/kg, 7.07 μg/kg, 9.4 μg/kg, or 12.5μg/kg, of the subject's body weight, administered three times a week,over a period of two weeks. In a specific aspect of this embodiment, theconjugate of the invention is administered every day for five days. Inother embodiments, the dosing may be repeated for multiple cycles,wherein the number of cycles chosen may or may not factor in themeasurement of anti-DT antibodies in the patient.

In another specific embodiment, the dosage of a conjugate of theinvention administered to a subject to prevent, treat, and/or managecancer in a patient is a unit dose of 0.1 μg to 20 μg, 0.1 μg to 15 μg,0.1 μg to 12 μg, 0.1 μg to 10 μg, 0.1 μg to 8 μg, 0.1 μg to 7 μg, 0.1 μgto 5 μg, 0.1 to 2.5 μg, 0.25 μg to 20 μg, 0.25 to 15 μg, 0.25 to 12 μg,0.25 to 10 μg, 0.25 to 8 μg, 0.25 μg to 7 μg, 0.25 μg to 5 μg, 0.5 μg to2.5 μg, 1 μg to 20 μg, 1 μg to 15 μg, 1 μg to 12 μg, 1 μg to 10 μg, 1 μgto 8 μg, 1 μg to 7 μg, 1 μg to 5 μg, or 1 μg to 2.5 μg.

In a specific embodiment, the dosage of a conjugate of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is in the range of 0.01 to 10 g/m², and more typically, in therange of 0.1 g/m² to 7.5 g/m², of the subject's body's surface area. Inone embodiment, the dosage administered to a subject is in the range of0.5 g/m² to 5 g/m², or 1 g/m² to 5 g/m² of the subject's body's surfacearea.

In other embodiments, the prophylactically and/or therapeuticallyeffective regimen comprises administering to a patient one or more dosesof an effective amount of a conjugate of the invention, wherein the doseof an effective amount achieves a plasma level of at least 0.1 μg/ml, atleast 0.5 μg/ml, at least 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml,at least 6 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20μg/ml, at least 25 μg/ml, at least 50 μg/ml, at least 100 μg/ml, atleast 125 μg/ml, at least 150 μg/ml, at least 175 μg/ml, at least 200μg/ml, at least 225 μg/ml, at least 250 μg/ml, at least 275 μg/ml, atleast 300 μg/ml, at least 325 μg/ml, at least 350 μg/ml, at least 375μg/ml, or at least 400 μg/ml of the compound of the invention.

In other embodiments, the prophylactically and/or therapeuticallyeffective regimen comprises administering to a patient a plurality ofdoses of an effective amount of a conjugate of the invention, whereinthe plurality of doses maintains a plasma level of at least 0.1 μg/ml,at least 0.15 μg/ml, at least 0.17 μg/ml, at least 0.2 μg/ml, at least0.23 μg/ml, at least 0.25 μg/ml, at least 0.3 μg/ml, at least 0.34μg/ml, at least 0.4 μg/ml, at least 0.45 μg/ml, at least 0.5 μg/ml, atleast 1 μg/ml, at least 2 μg/ml, at least 5 μg/ml, at least 6 μg/ml, atleast 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml,at least 50 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150μg/ml, at least 1751 g/ml, at least 200 μg/ml, at least 225 μg/ml, atleast 250 μg/ml, at least 275 μg/ml, at least 300 μg/ml, at least 325μg/ml, at least 350 μg/ml, at least 375 μg/ml, or at least 400 μg/ml ofthe compound of the invention for at least 1 month, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months,11 months, 12 months, 15 months, 18 months, or 24 months.

In another embodiment, the present invention encompassesprophylactically and/or therapeutically effective regimens wherein anamount of DT-IL3 conjugate is administered to a patient to achieveplasma levels of DT-IL3 conjugate in the range of at least 0.1 μg/ml toat least 20 μg/ml; at least 0.1 μg/ml to at least 50 μg/ml, at least 0.1μg/ml to at least 100 μg/ml, at least 0.1 μg/ml to at least 200 μg/ml,at least 0.1 μg/ml to at least 300 μg/ml, at least 0.1 μg/ml to at least400 μg/ml, at least 0.1 μg/ml to at least 500 μg/ml, at least 0.1 μg/mlto at least 600 μg/ml, at least 0.1 μg/ml to at least 700 μg/ml, or atleast 0.1 μg/ml to at least 800 μg/ml for at least 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, 15 months, 18 months, or 24 months.

In some embodiments, the response of a patient to a prophylacticallyand/or therapeutically effective regimen is monitored and the regimen ismaintained or adjusted based on a comparison to a reference point and/ormodel. In one embodiment, the regimen is maintained or adjusted based onthe monitoring of cancer cells. In another embodiment, the regimen ismaintained or adjusted based on the monitoring of cancer stem cells.

In specific embodiments, the patient's response to a treatment regimenis monitored through the collection and analysis of a sample from thepatient such as, but not limited to, a biological sample, e.g., thepatient's blood, bone marrow, normal tissue, or tumor biopsy. In oneembodiment, the reference point and/or model comprises pharmacokineticor immune response data from the patient undergoing therapy, wherein thedata is collected at an earlier time point (e.g., prior to receiving theregimen, as a baseline reference sample, or at an earlier time pointwhile receiving therapy). In another embodiment, the reference pointand/or model is from a healthy, non-cancer afflicted patient. In apreferred embodiment, the reference point is from a patient that hasachieved remission of cancer of the same type as the patient undergoingtreatment.

In certain embodiments, the response of a patient to a prophylacticallyand/or therapeutically effective regimen is monitored by measuring serumor plasma concentrations of a conjugate of the invention over time. Insome embodiments, the prophylactic and/or therapeutic regimen isadjusted as a result of the pharmacokinetic data obtained. For example,the frequency and/or dosage administered to the patient may be adjusted.In some embodiments, the response of a patient to a prophylacticallyand/or therapeutically effective regimen is monitored by assessing thepatient's immune response to the conjugate administered to the patient.In a specific embodiment, the patient's anti-diphtheria toxin (anti-DT)antibody titer is monitored. Several aspects of the regimen may bevaried based on the comparison including, but not limited to, the dosageand frequency of administration and the temporal regimen ofadministration.

In some embodiments, the titer of anti-DT antibodies in a patient ismeasured prior to administration of a conjugate of the invention. Thepretreatment anti-DT antibody titer may be considered in determining theeligibility of a patient to receive a conjugate of the invention, or theprophylactically and/or therapeutically effective regimen administeredto the patient. For example, a patient's anti-DT antibody titer maysuggest administering a conjugate of the invention at a particulardosage, at a particular frequency and/or for a certain period of time.

In some embodiments, the prophylactically and/or therapeuticallyeffective regimen comprises administration of a conjugate of theinvention in combination with one or more additional cancertherapeutics. See Section 5.3.2. Preferably, the dosages of the one ormore additional cancer therapeutics used in the combination therapy islower than those which have been or are currently being used to prevent,treat, and/or manage cancer. The recommended dosages of the one or moreadditional cancer therapeutics currently used for the prevention,treatment, and/or management of cancer can be obtained from anyreference in the art including, but not limited to, Hardman et al.,eds., Goodman & Gilman's The Pharmacological Basis Of Basis OfTherapeutics, 10th ed., Mc-craw-Hill, New York, 2001; Physician's DeskReference (60^(th) ed., 2006), which is incorporated herein by referencein its entirety.

The conjugate of the invention and the one or more additional cancertherapeutics can be administered separately, simultaneously, orsequentially. In various embodiments, the compound of the invention andthe additional cancer therapeutic are administered less than 5 minutesapart, less than 30 minutes apart, less than 1 hour apart, at about 1hour apart, at about 1 to about 2 hours apart, at about 2 hours to about3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In preferredembodiments, two or more cancer therapeutics are administered within thesame patient visit.

In certain embodiments, the conjugate of the invention and theadditional cancer therapeutic are cyclically administered. Cyclingtherapy involves the administration of one cancer therapeutic for aperiod of time, followed by the administration of a second cancertherapeutic for a period of time and repeating this sequentialadministration, i.e., the cycle, in order to reduce the development ofresistance to one or both of the cancer therapeutics, to avoid or reducethe side effects of one or both of the cancer therapeutics, and/or toimprove the efficacy of the therapies.

In a preferred embodiment, the cancer therapeutics are administeredconcurrently to a subject in separate compositions. The combinationcancer therapeutics of the invention may be administered to a subject bythe same or different routes of administration.

In a specific embodiment, cycling therapy involves the administration ofa first cancer therapeutic for a period of time, followed by theadministration of a second cancer therapeutic for a period of time,optionally, followed by the administration of a third cancer therapeuticfor a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the cancer therapeutics, to avoid or reduce theside effects of one of the cancer therapeutics, and/or to improve theefficacy of the cancer therapeutics.

When a conjugate of the invention and the additional cancer therapeuticare administered to a subject concurrently, the term “concurrently” isnot limited to the administration of the cancer therapeutics at exactlythe same time, but rather, it is meant that they are administered to asubject in a sequence and within a time interval such that they can acttogether (e.g., synergistically to provide an increased benefit than ifthey were administered otherwise). For example, the cancer therapeuticsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect, preferably in a synergistic fashion. Thecombination cancer therapeutics of the invention can be administeredseparately, in any appropriate form and by any suitable route. When thecomponents of the combination cancer therapeutics are not administeredin the same pharmaceutical composition, it is understood that they canbe administered in any order to a subject in need thereof. For example,a conjugate of the invention can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of theadditional cancer therapeutic, to a subject in need thereof. In variousembodiments, the cancer therapeutics are administered 1 minute apart, 10minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hoursapart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hoursto 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hoursapart, no more than 24 hours apart, or no more than 48 hours apart. Inone embodiment, the cancer therapeutics are administered within the sameoffice visit. In another embodiment, the combination cancer therapeuticsof the invention are administered at 1 minute to 24 hours apart.

5.3.2 Types of Diseases and Disorders

The present invention provides methods of treating or preventing ormanaging a disease or disorder characterized by cells expressing theIL-3 receptor beta subunit in humans by administering to humans in needof such treatment or prevention a pharmaceutical composition comprisingan amount of IL-3-diphtheria toxin conjugate of the invention effectiveto treat or prevent the disease or disorder. In certain embodiments, thedisease or disorder is not a hematologic cancer. In other embodiments,the disease or disorder is an allergic disease or disorder. In otherembodiments, the disease or disorder is an inflammatory disease ordisorder. In another embodiment, the disease or disorder is onecharacterized as affecting plasmacytoid dendritic cells (e.g., dentriticcell cancers such as NK blastic leukemia and CD4⁺ CD56⁺ dermatologicneoplasm). In certain embodiments, the subjects have acute myelogenousleukemia (AML). In certain other embodiments, the subjects havemyelodysplastic syndrome (MDS). In other embodiments, the subjects havechronic myelomonocytic leukemia (CMML), CML, ALL, hairy cell leukemia,Hodgkin's disease, or non-Hodgkin's lymphoma.

The present invention encompasses methods for preventing, treating,managing, and/or ameliorating an inflammatory disorder or one or moresymptoms thereof as an alternative to other conventional therapies. Inspecific embodiments, the patient being managed or treated in accordancewith the methods of the invention is refractory to other therapies or issusceptible to adverse reactions from such therapies. The patient may bea person with a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease,patients with broncho-pulmonary dysplasia, patients with congenitalheart disease, patients with cystic fibrosis, patients with acquired orcongenital heart disease, and patients suffering from an infection), aperson with impaired renal or liver function, the elderly, children,infants, infants born prematurely, persons with neuropsychiatricdisorders or those who take psychotropic drugs, persons with historiesof seizures, or persons on medication that would negatively interactwith conventional agents used to prevent, manage, treat, or ameliorate aviral respiratory infection or one or more symptoms thereof.

In an embodiment of the invention, diseases that are characterized byplasmacytoid dendritic cells, which cells demonstrate high expression ofthe alpha chain of the IL-3 receptor, are targeted. Such diseasesinclude, but are not limited to, HIV, herpes, CMV, autoimmune diseases,and cancers including but not limited to NK blastic lymphoma, dendriticcell cancer including plasmacytoid dendritic cell cancer, anddermatologic neoplasms.

Autoimmune Disorders

In certain embodiments, the invention provides a method of preventing,treating, managing, and/or ameliorating an autoimmune disorder or one ormore symptoms thereof, said method comprising administering to a subjectin need thereof a dose of an effective amount of one or morepharmaceutical compositions of the invention, wherein the cells involvedin such disorders express the interleukin-3 receptor beta subunit. Inautoimmune disorders, the immune system triggers an immune response andthe body's normally protective immune system causes damage to its owntissues by mistakenly attacking self. There are many differentautoimmune disorders which affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progress,destruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin.

Examples of autoimmune disorders that can be prevented, treated,managed, and/or ameliorated by the methods of the invention include, butare not limited to, adrenergic drug resistance, alopecia greata,ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune diseases of the adrenal gland, allergicencephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inflammatory eye disease, autoimmune neonatalthrombocytopenia, autoimmune neutropenia, autoimmune oophoritis andorchitis, autoimmune thrombocytopenia, autoimmune thyroiditis, Behcet'sdisease, bullous pemphigoid, cardiomyopathy, cardiotomy syndrome, celiacsprue-dermatitis, chronic active hepatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, dense depositdisease, discoid lupus, essential mixed cryoglobulinemia,fibromyalgia-fibromyositis, glomerulonephritis (e.g., IgA nephrophathy),gluten-sensitive enteropathy, Goodpasture's syndrome, Graves' disease,Guillain-Barre, hyperthyroidism (i.e., Hashimoto's thyroiditis),idiopathic pulmonary fibrosis, idiopathic Addison's disease, idiopathicthrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erythematosus, Ménière's disease, mixed connectivetissue disease, multiple sclerosis, Myasthenia Gravis, myocarditis, type1 or immune-mediated diabetes mellitus, neuritis, other endocrine glandfailure, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, Polyendocrinopathies, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, post-MI,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynauld's phenomenon, relapsing polychondritis,Reiter's syndrome, rheumatic heart disease, rheumatoid arthritis,sarcoidosis, scleroderma, Sjögren's syndrome, stiff-man syndrome,systemic lupus erythematosus, takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, urticaria, uveitis,Uveitis Opthalmia, vasculitides such as dermatitis herpetiformisvasculitis, vitiligo, and Wegener's granulomatosis.

Allergies

In certain embodiments, the invention provides a method of preventing,treating, managing, and/or ameliorating one or more allergic diseases orallergies or one or more symptoms thereof, wherein the cells involved insuch diseases or allergies express the interleukin-3 receptor betasubunit, said method comprising administering to a subject in needthereof a dose of an effective amount of one or more pharmaceuticalcompositions of the invention. Immune-mediated allergic(hypersensitivity) reactions are classified into four types (I-IV)according to the underlying mechanisms leading to the manifestation ofthe allergic symptoms. Type I allergic reactions are immediatehypersensitivity reactions characterized by IgE-mediated release ofvasoactive substances such as histamine from mast cells and basophils.Over hours, the mast cells and basophils release proinflammatorycytokines producing vasodilation, increased capillary permeability,glandular hypersecretion, smooth muscle spasm, and tissue infiltrationwith eosinophils and other inflammatory cells.

Type II allergic reactions are cytotoxic hypersensitivity reactions andinvolve IgG or IgM antibodies bound to cell surface antigens withsubsequent complement fixation. Certain cytotoxic cells, such as killerT cells or macrophages, are activated, bind to cells coated with IgG anddestroy the target cells. Type II reactions may result in cytolysis ortissue damage.

Type III reactions are immune-complex reactions resulting from depositsof circulating antigen-antibody immune complexes in blood vessels ortissues. Acute inflammation results from the immune-complex initiating asequence of events that results in polymorphonuclear cell migration andrelease of lysosomal proteolytic enzymes and permeability factors intissues.

Type IV reactions are delayed hypersensitivity reactions caused bysensitized T lymphocytes after contact with a specific antigen.Activated sensitized T lymphocytes cause immunologic injury by directtoxic effect or through release of lymphokines and other solublesubstances. The activated T lymphocytes may also release cytokines thataffect the activity of macrophages, neutrophils, and lymphoid killercells.

Allergic reactions can be immediate, late-phase, or chronic. Continuousor chronic exposure to an allergen can result in chronic allergicinflammation. Tissues of sites of chronic inflammation containeosinophils and T cells that release mediators that can cause tissuedamage, increased inflammation, and increased sensitivity.

Currently, allergic reactions are treated with drugs such asantihistamines, corticosteroids, vasodilators, bronchodilators,leukotriene inhibitors, and immunomodulators which attempt to alleviatethe symptoms associated with the allergic reaction.

Cancer

Any type of cancer in which the cancer stem cells or cancer cellsexpress the interleukin-3 receptor beta and/or alpha subunits can beprevented, treated, and/or managed in accordance with the invention.Non-limiting examples of cancers that can be prevented, treated, and/ormanaged in accordance with the invention include: leukemias, such as butnot limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias, such as, myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia leukemias andmyelodysplastic syndrome; chronic leukemias, such as but not limited to,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenström'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; dendritic cellcancer, including plasmacytoid dendritic cell cancer, NK blasticlymphoma (also known as cutaneous NK/T-cell lymphoma and agranular(CD4+/CD56+) dermatologic neoplasms); basophilic leukemia; bone andconnective tissue sarcomas such as but not limited to bone sarcoma,osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant celltumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to papillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

The prophylactically and/or therapeutically effective regimens are alsouseful in the treatment, prevention and/or management of a variety ofcancers or other abnormal proliferative diseases wherein the cells ofsuch diseases express the interleukin-3 receptor beta subunit, including(but not limited to) the following: carcinoma, including that of thebladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach,cervix, thyroid and skin; including squamous cell carcinoma;hematopoietic tumors of lymphoid lineage, including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, Tcell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloidlineage, including acute and chronic myelogenous leukemias andpromyelocytic leukemia; tumors of mesenchymal origin, includingfibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma,seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of thecentral and peripheral nervous system, including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and othertumors, including melanoma, xeroderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer and teratocarcinoma. In someembodiments, cancers associated with aberrations in apoptosis areprevented, treated and/or managed in accordance with the methods of theinvention. Such cancers may include, but not be limited to, follicularlymphomas, carcinomas with p53 mutations, hormone dependent tumors ofthe breast, prostate and ovary, and precancerous lesions such asfamilial adenomatous polyposis, and myelodysplastic syndromes. Inspecific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders of theskin, lung, liver, bone, brain, stomach, colon, breast, prostate,bladder, kidney, pancreas, ovary, and/or uterus are prevented, treatedand/or managed in accordance with the methods of the invention. In otherspecific embodiments, a sarcoma, melanoma, or leukemia is prevented,treated and/or managed in accordance with the methods of the invention.In certain embodiments, the subjects have acute myelogenous leukemia(AML). In certain other embodiments, the subjects have myelodysplasticsyndrome (MDS). In other embodiments, the subjects have chronicmyelomonocytic leukemia (CMML). In other specific embodiments,myelodysplastic syndrome is prevented, treated and/or managed inaccordance with the methods of the invention.

5.3.3 Target Patient Populations

In accordance with the invention, the pharmaceutical compositions of thepresent invention are administered to humans in need of inhibition ofcells that express the alpha subunit (in specific embodiments, the alphaand beta subunits) of interleukin-3 In certain embodiments, the growthof such cells is inhibited. In other embodiments, the conjugates of thepresent invention are administered to humans with diseases and disordersassociated with overexpression of the IL-3 receptor. In certainembodiments, the subject does have myeloid leukemia. In otherembodiments, the disease or disorder is an allergic disease or disorder.In some embodiments, the disease or disorder is an autoimmune disease.In certain embodiments, the subjects have acute myelogenous leukemia(AML). In certain other embodiments, the subjects have myelodysplasticsyndrome (MDS). In other embodiments, the subjects have chronicmyelomonocytic leukemia (CMML).

In accordance with the invention, pharmaceutical compositions of thepresent invention are administered to subjects developing, developed, orexpected to develop cancer (e.g., subjects with a genetic predispositionfor a particular type of cancer, subjects that have been exposed to acarcinogen, subjects with newly diagnosed cancer, subjects that havefailed treatment for cancer, subjects who have relapsed from cancer, orsubjects that are in remission from a particular cancer). Such subjectsmay or may not have been previously treated for cancer or may be inremission, relapsed, or may have failed treatment. Such patients mayalso have abnormal cytogenetics. The pharmaceutical compositions may beused as any line of cancer therapy, e.g., a first line, second line, orthird line of cancer therapy. In a specific embodiment, the subject toreceive or receiving a pharmaceutical composition of the invention isreceiving or has received other cancer therapies. In another embodimentthe subject to receive a pharmaceutical composition of the invention isreceiving other cancer therapies and pharmaceutical compositions of theinvention are administered to the subject before any adverse effects orintolerance of these other cancer therapies occurs. In an alternativeembodiment, the subject to receive or receiving a pharmaceuticalcomposition of the invention has not received or is not receiving othercancer therapies.

In a specific embodiment, the subject has been diagnosed with cancerusing techniques known to one of skill in the art including, but notlimited to, physical examination (e.g., prostate examination, breastexamination, lymph nodes examination, abdominal examination, skinsurveillance, general palpation), visual methods (e.g., colonoscopy,bronchoscopy, endoscopy), PAP smear analyses (cervical cancer), stoolguaiac analyses, blood tests (e.g., complete blood count (CBC) test,prostate specific antigen (PSA) test, carcinoembryonic antigen (CEA)test, cancer antigen (CA)-125 test, alpha-fetoprotein (AFP), liverfunction tests), karyotyping analyses, bone marrow analyses (e.g., incases of hematological malignancies), histology, flow cytometry,cytology, a sputum analysis and imaging methods (e.g., computedtomography (CT), magnetic resonance imaging (MRI), ultrasound, X-rayimaging, mammography, PET scans, radionuclide scans, bone scans).Subjects may or may not have been previously treated for cancer.

In one embodiment, a pharmaceutical composition of the invention isadministered to a subject that is undergoing or has undergone surgery toremove a tumor neoplasm. In a specific embodiment, a pharmaceuticalcomposition of the invention is administered to a subject concurrentlyor following surgery to remove a tumor or neoplasm. In anotherembodiment, a pharmaceutical composition of the invention isadministered to a subject before surgery to remove a tumor or neoplasmand, in some embodiments, during and/or after surgery.

In one embodiment, a pharmaceutical composition of the invention isadministered to a subject after a course of therapy with the goal ofkilling cancer cells. In some embodiments, the course of therapyinvolves the administration of bolus doses of chemotherapeutic agentsand/or bolus doses of radiation therapy. In a specific embodiment, apharmaceutical composition of the invention is administered to a subjectafter the subject has received a course of therapy involving a dosewhich is at, or is below, the maximum tolerated dose or the no observedadverse effect level doses of one or more chemotherapeutic agents and/orradiation therapy.

In certain embodiments, a pharmaceutical composition of the invention isadministered to a subject as an alternative to chemotherapy, radiationtherapy, hormonal therapy, surgery, small molecule therapy,anti-angiogenic therapy, differentiation therapy, epigenetic therapy,radioimmunotherapy, targeted therapy, and/or biological therapyincluding immunotherapy where the therapy has proven or may prove tootoxic, i.e., results in unacceptable or unbearable side effects for thesubject. In some embodiments, a prophylactically and/or therapeuticallyeffective regimen is administered to a subject that is susceptible toadverse reactions from other cancer therapies. The subject may, e.g.,have a suppressed immune system (e.g., post-operative patients,chemotherapy patients, and patients with immunodeficiency disease), havean impaired renal or liver function, be elderly, be a child, be aninfant, have a neuropsychiatric disorder, take a psychotropic drug, havea history of seizures, or be on medication that would negativelyinteract with the cancer therapies.

In a specific embodiment, a pharmaceutical composition of the inventionis administered to subjects that will, are or have radiation therapy.Among these subjects are those that have received chemotherapy, hormonaltherapy, small molecule therapy, anti-angiogenic therapy,differentiation therapy, targeted therapy, radioimmunotherapy,epigenetic therapy, and/or biological therapy, including immunotherapyas well as those who have undergone surgery.

In another embodiment, a pharmaceutical composition of the invention isadministered to subjects that will, are, or have received hormonaltherapy and/or biological therapy, including immunotherapy. Among thesesubjects are those that have received chemotherapy, small moleculetherapy, anti-angiogenic therapy, differentiation therapy, targetedtherapy, radioimmunotherapy, epigenetic therapy, and/or radiationtherapy as well as those who have undergone surgery.

In certain embodiments, a pharmaceutical composition of the invention isadministered to a subject refractory to one or more therapies. In oneembodiment, that a cancer is refractory to a therapy means that at leastsome significant portion of the cancer cells are not killed or theircell division is not arrested. The determination of whether the cancercells are refractory can be made either in vivo or in vitro by anymethod known in the art for assaying the effectiveness of a therapy oncancer cells, using the art-accepted meanings of “refractory” in such acontext. In various embodiments, a cancer is refractory where the amountof cancer cells has not been significantly reduced, or has increased. Inother embodiments, that a cancer is refractory means that at least somesignificant portion of cancer stem cells are not killed or their celldivision arrested. The determination of whether the cancer stem cellsare refractory can be made either in vivo or in vitro by any methodsknown in the art or described herein.

In some embodiments, a pharmaceutical composition of the invention isadministered to reverse the resistance to, or increase the sensitivityof cancer cells to certain hormonal, radiation and chemotherapeuticagents thereby resensitizing the cancer cells to one or more of theseagents, which can then be administered (or continue to be administered)to treat or manage cancer, including to prevent metastasis. In aspecific embodiment, the regimens of the invention are administered topatients with increased levels of the cytokine IL-6, which has beenassociated with the development of cancer cell resistance to differenttreatment regimens, such as chemotherapy and hormonal therapy.

In some embodiments, a pharmaceutical composition of the invention isadministered to a subject with a mean absolute lymphocyte count of atleast approximately 400 cells/mm³, at least 500 cells/mm³, at leastapproximately 600 cells/mm³, at least approximately 700 cells/mm³, atleast approximately 800 cells/mm³, at least approximately 900 cells/mm³,at least approximately 1000 cells/mm³, at least approximately 1100cells/mm³, at least approximately 1200 cells/mm³. In other embodiments,a prophylactically and/or therapeutically effective regimen of theinvention is administered to a subject with a mean absolute lymphocytecount of approximately 400 cells/mm³ to approximately 1200 cells/mm³,approximately 500 cells/mm³ to approximately 1200 cells/mm³,approximately 600 cells/mm³ to approximately 1200 cells/mm³,approximately 700 cells/mm³ to approximately 1200 cells/mm³,approximately 800 cells/mm³ to approximately 1200 cells/mm³,approximately 900 cells/mm³ to approximately 1200 cells/mm³,approximately 1000 cells/mm³ to approximately 1200 cells/mm³. In a morespecific embodiment, the regimen results in a mean absolute lymphocytecount of at least approximately 400 cells/mm³.

In some embodiments, a pharmaceutical composition of the invention isadministered to a subject that is in remission. In a specificembodiment, the subject has no detectable cancer, i.e., no cancer isdetectable using a conventional method described herein (e.g., MRI) orknown to one of skill in the art. In another embodiment, apharmaceutical composition of the invention is administered to a patientthat does not have a detectable immune response to diphtheria toxin. Ina preferred embodiment, the immune response is detected by ELISA.

5.3.4 Combination Therapies

The present invention also provides methods for preventing, treating,and/or managing cancer, the methods comprising administering to apatient (e.g., a human patient) in need thereof, a prophylacticallyand/or a therapeutically effective regimen, the regimen comprisingadministering to the patient a pharmaceutical composition of theinvention and one or more additional therapies, said additional therapynot being a conjugate of the invention. In a specific embodiment, thecombination therapies of the invention comprise a pharmaceuticalcomposition in accordance with the invention and at least one othertherapy that has the same mechanism of action as said conjugate. Inanother specific embodiment, the combination therapies of the inventioncomprise a pharmaceutical composition identified in accordance with themethods of the invention and at least one other therapy (e.g.,prophylactic or therapeutic agent) which has a different mechanism ofaction than said conjugate. The pharmaceutical composition of theinvention and the additional therapy can be administered separately,concurrently, or sequentially. The combination of agents can actadditively or synergistically. The combination therapies of the presentinvention reduce the side effects associated with the therapies (e.g.,prophylactic or therapeutic agents).

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, or is currently being used for the prevention, treatment,and/or management of cancer can be used in compositions and methods ofthe invention. Therapies (e.g., therapeutic or prophylactic agents)include, but are not limited to, peptides, polypeptides, antibodies,conjugates, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapies include chemotherapy,radiation therapy, hormonal therapy, surgery, small molecule therapy,anti-angiogenic therapy, differentiation therapy, epigenetic therapy,radioimmunotherapy, targeted therapy, and/or biological therapyincluding immunotherapy. In certain embodiments, a prophylacticallyand/or therapeutically effective regimen of the invention comprises theadministration of a combination of therapies.

Examples of cancer therapies include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; histone deacetylase inhibitors (HDACs) gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; imatinib mesylate (Gleevec, Glivec); interleukin II(including recombinant interleukin II, or rIL2), interferon alpha-2a;interferon alpha-2b; interferon alpha-n1; interferon alpha-n3;interferon beta-I a; interferon gamma-I b; iproplatin; irinotecanhydrochloride; lanreotide acetate; lenalidomide (Revlimid); letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; anti-CD2 antibodies (e.g., siplizumab(MedImmune Inc.; International Publication No. WO 02/098370, which isincorporated herein by reference in its entirety)); megestrol acetate;melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;nogalamycin; ormaplatin; oxaliplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; VITAXIN™ (see U.S. Patent Pub. No. US2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering Integrinαvβ3 Antagonists in Combination With Other Prophylactic or TherapeuticAgents”); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

A non-limiting list of compounds that could be used to target cancerstem cells includes inhibitors of interleukin-3 receptor (IL-3R) andCD123 (including peptides, peptide-conjugates, antibodies,antibody-conjugates, antibody fragments, and antibodyfragment-conjugates that target IL-3R or CD123); cantharidin;norcantharidin and analogs and derivatives thereof; Notch pathwayinhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodelglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti CD20antibodies and conjugates (e.g. Rituxan, Bexxar, Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-1; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcl-2; antibodies to CLL-1; and furin inhibitors (such ascucurbitacins).

An additional non-limiting list of compounds that could also be used totarget cancer stem cells includes i) antibodies, antibody fragments, andproteins that are either naked or conjugated to a therapeutic moietythat target certain cell surface targets on cancer stem cells, or ii)small molecules known in the art including ones that can be furtheroptimized (e.g. via chemistry) or identified via a cancer stemcell-based screen (e.g. such as one that would determine whether acompound impairs proliferation or viability of a cancer stem cellthrough standard methods, the cell surface and intracellular targetsincluding (not meant to be exhaustive) are: Rex 1 (Zfp42), CTGF, ActivinA, Wnt, FGF-2, HIF-1, AP-2gamma, Bmi-1, nucleostemin, hiwi, Moz-TIF2,Nanog, beta-arrestin-2, Oct-4, Sox2, stella, GDF3, RUNX3, EBAF, TDGF-1,nodal, ZFPY, PTNE, Evi-1, Pax3, Mcl-1, c-kit, Lex-1, Zfx, lactadherin,aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2, CD26, Gremlin,and FoxC2.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an immunomodulatory agent. Non-limitingexamples of immunomodulatory agents include proteinaceous agents such ascytokines, peptide mimetics, and antibodies (e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments orepitope binding fragments), nucleic acid molecules (e.g., antisensenucleic acid molecules and triple helices), small molecules, organiccompounds, and inorganic compounds. In particular, immunomodulatoryagents include, but are not limited to, methotrexate, leflunomide,cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline,azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone(MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin(sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an anti-angiogenic agent. Non-limitingexamples of anti-angiogenic agents include proteins, polypeptides,peptides, conjugates, antibodies (e.g., human, humanized, chimeric,monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F(ab)2 fragments, andantigen-binding fragments thereof) such as antibodies that specificallybind to TNF-α, nucleic acid molecules (e.g., antisense molecules ortriple helices), organic molecules, inorganic molecules, and smallmolecules that reduce or inhibit angiogenesis. Other examples ofanti-angiogenic agents can be found, e.g., in U.S. Publication No.2005/0002934 A1 at paragraphs 277-282, which is incorporated byreference in its entirety. In other embodiments, the therapy(ies) usedin accordance with the invention is not an anti-angiogenic agent.

In some embodiments, the therapy(ies) used in combination with acompound of the invention is an anti-inflammatory agent. Non-limitingexamples of anti-inflammatory agents include any anti-inflammatoryagent, including agents useful in therapies for inflammatory disorders,well-known to one of skill in the art. Non-limiting examples ofanti-inflammatory agents include non-steroidal anti-inflammatory drugs(NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (e.g.,atropine sulfate, atropine methylnitrate, and ipratropium bromide(ATROVENT™)), beta2-agonists (e.g., abuterol (VENTOLIN™ and PROVENTIL™),bitolterol (TORNALATE™), levalbuterol (XOPONEX™), metaproterenol(ALUPENT™), pirbuterol (MAXAIR™), terbutlaine (BRETHAIRE™ andBRETHINE™), albuterol (PROVENTIL™, REPETABS™, and VOLMAX™), formoterol(FORADIL AEROLIZER™), and salmeterol (SEREVENT™ and SEREVENT DISKUS™)),and methylxanthines (e.g., theophylline (UNIPHYL™, THEO-DUR™, SLO-BID™,AND TEHO-42™)). Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), corticosteroids (e.g., methylprednisolone (MEDROL™)),cortisone, hydrocortisone, prednisone (PREDNISONE™ and DELTASONE™),prednisolone (PRELONE™ and PEDIAPRED™), triamcinolone, azulfidine, andinhibitors of eicosanoids (e.g., prostaglandins, thromboxanes, andleukotrienes. Other examples of anti-inflammatory agents can be found,e.g., in U.S. Publication No. 005/0002934 A1 at paragraphs 290-294,which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) used in accordance with the invention isnot an anti-inflammatory agent.

In certain embodiments, the therapy(ies) used is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, cholormbucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine).

The invention includes the use of agents that target cancer stem cellsin combination with a compound of the invention. In some embodiments,the agent used is an agent that binds to a marker, e.g., antigen oncancer stem cells. In a specific embodiment, the agent binds to anantigen that is expressed at a greater level on cancer stem cells thanon normal stem cells. In a specific embodiment, the agent bindsspecifically to a cancer stem cell antigen. In other embodiments, thetherapy(ies) used in accordance with the invention is an agent thatbinds to a marker on cancer stem cells. Non-limiting examples ofantigens on cancer stem cells that can be used to target cancer stemcells include CD34+/CD38−, CD34+/CD38−/CD123+, CD44+/CD24−, CD133+,CD34+/CD10−/CD19−, CD138−/CD34−/CD19+, CD20+, CD133+/RC2+, andCD44+/α2β1hi/C133+. In one embodiment, the agent that binds to a markeron cancer stem cells is an antibody. In another embodiment, the agentthat binds to a marker on cancer stem cells is a ligand. In certainembodiments, the antibody or ligand is attached directly or indirectlyto a therapeutic moiety. Non-limiting examples of therapeutic moietiesinclude, but are not limited to alkylating agents, anti-metabolites,plant alkaloids, cytotoxic agents, chemotherapeutic agents (e.g., asteroid, cytosine arabinoside, fluoruracil, methotrexate, aminopterin,mitomycin C, demecolcine, etoposide, mithramycin, calicheamicin,CC-1065, chlorambucil or melphalan), radionuclides, therapeutic enzymes,cytokines, toxins including plant-derived toxins, fungus-derived toxins,bacteria-derived toxin (e.g., deglycosylated ricin A chain, a ribosomeinactivating protein, alpha-sarcin, aspergillin, restirictocin, aribonuclease, a diphtheria toxin, Pseudomonas exotoxin, a bactericalendotoxin or the lipid A moiety of a bacterial endotoxin), growthmodulators and RNase.

For example, in a specific embodiment, the agent binds specifically tothe IL-3 Receptor (IL-3R). In some embodiments, the agent that binds tothe IL-3R is an antibody or an antibody fragment that is specific forIL-3R. In some embodiments, the antibody or antibody fragment isconjugated either chemically or via recombinant technology to atherapeutic moiety (e.g., a chemotherapeutic agent, a plant-, fungus- orbacteria-derived toxin, a radionuclide) using a linking agent to effecta cell killing response. In certain embodiments, the antibody,antibody-conjugate, antibody fragment, or antibody fragment-conjugatebinds to the α-subunit of IL-3R (i.e., the CD123 antigen). In otherembodiments, the antibody, antibody-conjugate, antibody fragment, orantibody fragment-conjugate binds to the IL-3R, containing both the αand β subunits. Methods for preparing antibodies to IL-3R and mimeticsof antibodies to IL-3R are described in U.S. Pat. No. 6,733,743 B2,which is incorporated herein by reference in its entirety.

In certain embodiments, antibodies or fragments that bind to a marker oncancer stem cells are substantially non-immunogenic in the treatedsubject. Non-immunogenic antibodies include, but are not limited to,chimerized antibodies, humanized antibodies, and antibodies from thesame species as the subject receiving the therapy. Antibodies orfragments that bind to markers in cancer stem cells can be producedusing techniques known in the art. See, for example, paragraphs 539-573of U.S. Publication No. 2005/0002934 A1, which is incorporated byreference in its entirety.

The invention includes the use of agents that target cancer stem cells.In certain embodiments, the agent acts alone. In other embodiments, theagent is attached directly or indirectly to another therapeutic moiety.Non-limiting examples of therapeutic moieties include, but are notlimited to, therapeutic enzymes, chemotherapeutic agents, cytokines,radionuclides, toxins, RNase, and antimetabolites. In some embodiments,the agent used is an agent that binds to a marker, e.g., an antigen on acancer stem cell. In a specific embodiment, the agent binds to anantigen that is expressed at a greater level on cancer stem cells thanon normal stem cells. In a specific embodiment, the agent bindsspecifically to a cancer stem cell antigen that is not a normal stemcell. In other embodiments, the therapy(ies) is an agent that binds to amarker on cancer stem cells. In one embodiment, the agent that binds toa marker on cancer stem cells is an antibody, an antibody fragment, anantibody conjugated to a therapeutic moiety, or an antibody fragmentconjugated to a therapeutic moiety.

In some embodiments, a compound of the invention is used in combinationwith radiation therapy comprising the use of X-rays, gamma rays andother sources of radiation to destroy cancer stem cells and/or cancercells. In specific embodiments, the radiation therapy is administered asexternal beam radiation or teletherapy, wherein the radiation isdirected from a remote source. In other embodiments, the radiationtherapy is administered as internal therapy or brachytherapy wherein aradioactive source is placed inside the body close to cancer stem cells,cancer cells and/or a tumor mass.

In some embodiments, the therapy used is a proliferation-based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra.

Currently available cancer therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006). In accordance with the present invention, the dosages andfrequency of administration of chemotherapeutic agents are describedsupra.

5.4 Methods of Monitoring Cancer Stem Cells

As part of the prophylactically effective and/or therapeuticallyeffective regimens of the invention, the cancer stem cell population canbe monitored to assess the efficacy of a therapy as well as to determineprognosis of a subject with cancer or the efficacy of a therapeuticallyor prophylactically effective regimen. In certain embodiments of theprophylactically effective and/or therapeutically effective therapies orregimens of the invention, the therapies or regimens result in astabilization or reduction in the cancer stem cell population in thepatient. In one embodiment, the subject undergoing the regimen ismonitored to assess whether the regimen has resulted in a stabilizationor reduction in the cancer stem cell population in the subject.

In some embodiments, the amount of cancer stem cells in a subject isdetermined using a technique well-known to one of skill in the art ordescribed in Section 5.7 below.

In accordance with the invention, cancer stem cells comprise a uniquesubpopulation (often 0.1-10% or so) of a tumor that, in contrast to theremaining 90% or so of the tumor (i.e., the tumor bulk), are relativelymore tumorigenic and relatively more slow-growing or quiescent. Giventhat conventional therapies and regimens have, in large part, beendesigned to attack rapidly proliferating cells (i.e., those cancer cellsthat comprise the tumor bulk) slower growing cancer stem cells may berelatively more resistant than faster growing tumor bulk to conventionaltherapies and regimens. This would explain another reason for thefailure of standard oncology treatment regimens to ensure long-termbenefit in most patients with advanced stage cancers. In a specificembodiment, a cancer stem cell(s) is the founder cell of a tumor (i.e.,it is the progenitor of cancer cells). In some embodiments, a cancerstem cell(s) has one, two, three, or more or all of the followingcharacteristics or properties: (i) can harbor the ability to initiate atumor and/or to perpetuate tumor growth, (ii) can be generallyrelatively less mutated than the bulk of a tumor (e.g. due to slowergrowth and thus fewer DNA replication-dependent errors, improved DNArepair, and/or epigenetic/non-mutagenic changes contributing to theirmalignancy), (iii) can have many features of a normal stem cell(s)(e.g., similar cell surface antigen and/or intracellular expressionprofile, self-renewal programs, multi-drug resistance, an immaturephenotype, etc., characteristic of normal stem cells) and may be derivedfrom a normal stem cell(s), (iv) can be potentially responsive to itsmicroenvironment (e.g., the cancer stem cells may be capable of beinginduced to differentiate and/or divide asymmetrically), (v) can be thesource of metastases, (vi) can be slow-growing or quiescent, (vii) canbe symmetrically-dividing, (viii) can be tumorigenic (e.g. as determinedby NOD/SCID implantation experiments), (ix) can be relatively resistantto traditional therapies (i.e. chemoresistant), and (x) can comprise asubpopulation of a tumor (e.g. relative to the tumor bulk).

In other embodiments, the amount of cancer stem cells in a sample from asubject is determined/assessed using a technique described herein orwell-known to one of skill in the art. Such samples include, but are notlimited to, biological samples and samples derived from a biologicalsample. In certain embodiments, in addition to the biological sampleitself or in addition to material derived from the biological samplesuch as cells, the sample used in the methods of this inventioncomprises added water, salts, glycerin, glucose, an antimicrobial agent,paraffin, a chemical stabilizing agent, heparin, an anticoagulant, or abuffering agent. In certain embodiments, the biological sample is blood,serum, urine, bone marrow or interstitial fluid. In another embodiment,the sample is a tissue sample. In a particular embodiment, the tissuesample is breast, brain, skin, colon, lung, liver, ovarian, pancreatic,prostate, renal, bone or skin tissue. In a specific embodiment, thetissue sample is a biopsy of normal or tumor tissue. The amount ofbiological sample taken from the subject will vary according to the typeof biological sample and the method of detection to be employed. In aparticular embodiment, the biological sample is blood, serum, urine, orbone marrow and the amount of blood, serum, urine, or bone marrow takenfrom the subject is 0.1 ml, 0.5 ml, 1 ml, 5 ml, 8 ml, 10 ml or more. Inanother embodiment, the biological sample is a tissue and the amount oftissue taken from the subject is less than 10 milligrams, less than 25milligrams, less than 50 milligrams, less than 1 gram, less than 5grams, less than 10 grams, less than 50 grams, or less than 100 grams.

In accordance with the methods of the invention, a sample derived from abiological sample is one in which the biological sample has beensubjected to one or more pretreatment steps prior to the detectionand/or measurement of the cancer stem cell population in the sample. Incertain embodiments, a biological fluid is pretreated by centrifugation,filtration, precipitation, dialysis, or chromatography, or by acombination of such pretreatment steps. In other embodiments, a tissuesample is pretreated by freezing, chemical fixation, paraffin embedding,dehydration, permeablization, or homogenization followed bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In certain embodiments,the sample is pretreated by removing cells other than stem cells orcancer stem cells from the sample, or removing debris from the sampleprior to the determination of the amount of cancer stem cells in thesample according to the methods of the invention.

The samples for use in the methods of this invention may be taken fromany animal subject, preferably mammal, most preferably a human. Thesubject from which a sample is obtained and utilized in accordance withthe methods of this invention includes, without limitation, anasymptomatic subject, a subject manifesting or exhibiting 1, 2, 3, 4, ormore symptoms of cancer, a subject clinically diagnosed as havingcancer, a subject predisposed to cancer, a subject suspected of havingcancer, a subject undergoing therapy for cancer, a subject that has beenmedically determined to be free of cancer (e.g., following therapy forthe cancer), a subject that is managing cancer, or a subject that hasnot been diagnosed with cancer. In certain embodiments, the term “has nodetectable cancer,” as used herein, refers to a subject or subjects inwhich no cancer is detectable using a conventional method describedherein (e.g., MRI) or known to one of skill in the art. In otherembodiments, the term refers to a subject or subjects free from anydisorder.

In certain embodiments, the amount of cancer stem cells in a subject ora sample from a subject assessed prior to therapy or regimen (e.g. atbaseline) or at least 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 16, 18, 20, 30,60, 90 days, 6 months, 9 months, 12 months, >12 months after the subjectbegins receiving the therapy or regimen. In certain embodiments, theamount of cancer stem cells is assessed after a certain number of doses(e.g., after 2, 5, 10, 20, 30, or more doses of a therapy). In otherembodiments, the amount of cancer stem cells is assessed after 1 week, 2weeks, 1 month, 2 months, 1 year, 2 years, 3 years, 4 years, or moreafter receiving one or more therapies.

In certain embodiments, a positive or negative control sample is asample that is obtained or derived from a corresponding tissue orbiological fluid as the sample to be analyzed in accordance with themethods of the invention. This sample may come from the same patient ordifferent persons and at the same or different time points.

For clarity of disclosure, and not by way of limitation, the followingpertains to analysis of a blood sample from a patient. However, as oneskilled in the art will appreciate, the assays and techniques describedherein can be applied to other types of patient samples, including abody fluid (e.g. blood, bone marrow, plasma, urine, bile, asciticfluid), a tissue sample suspected of containing material derived from acancer (e.g. a biopsy) or homogenate thereof. The amount of sample to becollected will vary with the particular type of sample and method ofdetermining the amount of cancer stem cells used and will be an amountsufficient to detect the cancer stem cells in the sample.

A sample of blood may be obtained from a patient having differentdevelopmental or disease stages. Blood may be drawn from a subject fromany part of the body (e.g., a finger, a hand, a wrist, an arm, a leg, afoot, an ankle, a stomach, and a neck) using techniques known to one ofskill in the art, in particular methods of phlebotomy known in the art.In a specific embodiment, venous blood is obtained from a subject andutilized in accordance with the methods of the invention. In anotherembodiment, arterial blood is obtained and utilized in accordance withthe methods of the invention. The composition of venous blood variesaccording to the metabolic needs of the area of the body it isservicing. In contrast, the composition of arterial blood is consistentthroughout the body. For routine blood tests, venous blood is generallyused.

The amount of blood collected will vary depending upon the site ofcollection, the amount required for a method of the invention, and thecomfort of the subject. In some embodiments, any amount of blood iscollected that is sufficient to detect the amount or amount of cancerstem cells. In a specific embodiment, 1 cc or more of blood is collectedfrom a subject.

The amount of cancer stem cells in a sample can be expressed as thepercentage of, e.g., overall cells, overall cancer cells or overall stemcells in the sample, or quantitated relative to area (e.g. cells perhigh power field), or volume (e.g. cells per ml), or architecture (e.g.cells per bone spicule in a bone marrow specimen).

In some embodiments, the sample may be a blood sample, bone marrowsample, or a tissue/tumor biopsy sample, wherein the amount of cancerstem cells per unit of volume (e.g., 1 ml) or other measured unit (e.g.,per unit field in the case of a histological analysis) is quantitated.In certain embodiments, the cancer stem cell population is determined asa portion (e.g., a percentage) of the cancerous cells present in theblood or bone marrow or tissue/tumor biopsy sample or as a subset of thecancerous cells present in the blood or bone marrow or tissue/tumorbiopsy sample. The cancer stem cell population, in other embodiments,can be determined as a portion (e.g., percentage) of the total cells. Inyet other embodiments, the cancer stem cell population is determined asa portion (e.g., a percentage) of the total stem cells present in theblood sample.

In other embodiments, the sample from the patient is a tissue sample(e.g., a biopsy from a subject with or suspected of having canceroustissue), where the amount of cancer stem cells can be measured, forexample, by immunohistochemistry or flow cytometry, or on the basis ofthe amount of cancer stem cells per unit area, volume, or weight of thetissue. In certain embodiments, the cancer stem cell population isdetermined as a portion (e.g., a percentage) of the cancerous cellspresent in the tissue sample or as a subset of the cancerous cellspresent in the tissue sample. In yet other embodiments, the cancerousstem cell population is determined as a portion (e.g., a percentage) ofthe overall cells or stem cells in the tissue sample.

The amount of cancer stem cells in a test sample can be compared withthe amount of cancer stem cells in a reference sample(s) to assess theefficacy of the regimen. In one embodiment, the reference sample is asample obtained from the subject undergoing therapy at an earlier timepoint (e.g., prior to receiving the regimen as a baseline referencesample, or at an earlier time point while receiving the therapy). Inthis embodiment, the therapy desirably results in a decrease in theamount of cancer stem cells in the test sample as compared with thereference sample. In another embodiment, the reference sample isobtained from a healthy, subject that has no detectable cancer, or froma patient that is in remission for the same type of cancer. In thisembodiment, the therapy desirably results in the test sample having anequal amount of cancer stem cells, or less than the amount of cancerstem cells than are detected in the reference sample.

In other embodiments, the cancer stem cell population in a test samplecan be compared with a predetermined reference range and/or a previouslydetected amount of cancer stem cells determined for the subject to gaugethe subject's response to the regimens described herein. In a specificembodiment, a stabilization or reduction in the amount of cancer stemcells relative to a predetermined reference range and/or earlier(previously detected) cancer stem cell amount determined for the subjectindicates an improvement in the subject's prognosis or a positiveresponse to the regimen, whereas an increase relative to thepredetermined reference range and/or earlier cancer stem cell amountindicates the same or worse prognosis, and/or a failure to respond tothe regimen. The cancer stem cell amount can be used in conjunction withother measures to assess the prognosis of the subject and/or theefficacy of the regimen. In a specific embodiment, the predeterminedreference range is based on the amount of cancer stem cells obtainedfrom a patient or population(s) of patients suffering from the same typeof cancer as the patient undergoing the therapy.

Generally, since stem cell antigens can be present on both cancer stemcells and normal stem cells, a sample from the cancer-afflicted patientwill have a higher stem cell count than a sample from a healthy subjectwith no detectable cancer, due to the presence of the cancer stem cells.The therapy will desirably result in a cancer stem cell count for thetest sample (e.g., the sample from the patient undergoing therapy) thatdecreases and becomes increasingly closer to the stem cell count in areference sample that is a sample from a healthy subject with nodetectable cancer by a conventional method.

If the reduction in amount of cancer stem cells is determined to beinadequate upon comparing the amount of cancer stem cells in the samplefrom the subject undergoing the regimen with the reference sample, thenthe medical practitioner has a number of possible options to adjust theregimen. For instance, the medical practitioner can then increase eitherthe dosage or intensity of the therapy administered, the frequency ofthe administration, the duration of administration, combine the therapywith another therapy(ies), change the management altogether includinghalting therapy, or any combination thereof.

In certain embodiments, the dosage, frequency and/or duration ofadministration of a therapy is modified as a result of the change in theamount of cancer stem cells detected in or from the treated patient. Forexample, if a subject receiving therapy for leukemia has a cancer stemcell measurement of 2.5% of his tumor prior to therapy and 5% after 6weeks of therapy, then the therapy or regimen may be altered or stoppedbecause the increase in the percentage of cancer stem cells indicatesthat the therapy or regimen is not optimal. Alternatively, if anothersubject with leukemia has a cancer stem cell measurement of 2.5% of histumor prior to therapy and 1% after 6 weeks of therapy, then the therapyor regimen may be continued because the decrease in the percentage ofcancer stem cells indicates that the therapy or regimen is effective.

The amount of cancer stem cells can be monitored/assessed using standardtechniques known to one of skill in the art. Cancer stem cells can bemonitored by, e.g., obtaining a sample, such as a tissue/tumor sample,blood sample or a bone marrow sample, from a subject and detectingcancer stem cells in the sample. The amount of cancer stem cells in asample (which may be expressed as percentages of, e.g., overall cells oroverall cancer cells) can be assessed by detecting the expression ofantigens on cancer stem cells. Techniques known to those skilled in theart can be used for measuring these activities. Antigen expression canbe assayed, for example, by immunoassays including, but not limited to,western blots, immunohistochemistry, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, immunofluorescence, protein A immunoassays, flowcytometry, and FACS analysis. In such circumstances, the amount ofcancer stem cells in a test sample from a subject may be determined bycomparing the results to the amount of stem cells in a reference sample(e.g., a sample from a subject who has no detectable cancer) or to apredetermined reference range, or to the patient him/herself at anearlier time point (e.g. prior to, or during therapy).

In a specific embodiment, the cancer stem cell population in a samplefrom a patient is determined by flow cytometry. This method exploits thedifferential expression of certain surface markers on cancer stem cellsrelative to the bulk of the tumor. Labeled antibodies (e.g., fluorescentantibodies) can be used to react with the cells in the sample, and thecells are subsequently sorted by FACS methods. In some embodiments, acombination of cell surface markers are utilized in order to determinethe amount of cancer stem cells in the sample. For example, bothpositive and negative cell sorting may be used to assess the amount ofcancer stem cells in the sample. Cancer stem cells for specific tumortypes can be determined by assessing the expression of markers on cancerstem cells. In certain embodiments, the tumors harbor cancer stem cellsand their associated markers as set forth in Table 2 below, whichprovides a non-limiting list of cancer stem cell phenotypes associatedwith various types of cancer.

TABLE 2 Tumor Cancer Stem Cell Phenotype Leukemia (AML) CD34+/CD38−Breast CD44+/CD24− Brain CD133+ Leukemia (ALL) CD34+/CD10−/CD19− OvarianCD44+/CD24− Multiple Myeloma CD138−/CD34−/CD19+ Chronic myelogenousleukemia CD34+/CD38− Melanoma CD20+ Ependymoma CD133+/RC2+ ProstateCD44+/α₂β₁ ^(hi)/CD133+

Additional cancer stem cell markers include, but are not limited to,CD123, CLL-1, combinations of SLAMs (signaling lymphocyte activationmolecule family receptors; see Yilmaz et al., “SLAM family markers areconserved among hematopoietic stem cells from old and reconstituted miceand markedly increase their purity,” Hematopoiesis 107: 924-930 (2006)),such as CD150, CD244, and CD48, and those markers disclosed in U.S. Pat.No. 6,004,528 to Bergstein, in pending U.S. patent application Ser. No.09/468,286, and in U.S. Patent Application Publication Nos.2006/0083682, 2007/0036800, 2007/0036801, 2007/0036802, 2007/0041984,2007/0036803, and 2007/0036804, each of which are incorporated herein byreference in their entirety. See, e.g., Table 1 of U.S. Pat. No.6,004,528 and Tables 1, 2, and 3 of U.S. patent application Ser. No.09/468,286 and U.S. Patent Application Publication Nos. 2006/0083682,2007/0036800, 2007/0036801, 2007/0036802, 2007/0041984, 2007/0036803,and 2007/0036804.

In a specific embodiment the cancer stem population in a sample, e.g., atissue sample, such as a solid tumor biopsy, is determined usingimmunohistochemistry techniques. This method exploits the differentialexpression of certain surface markers on cancer stem cells relative tothe bulk of the tumor. Labeled antibodies (e.g., fluorescent antibodies)can be used to react with the cells in the sample, and the tissue issubsequently stained. In some embodiments, a combination of certain cellsurface markers are utilized in order to determine the amount of cancerstem cells in the sample. Cancer stem cells for specific tumor types canbe determined by assessing the expression of certain markers that arespecific to cancer stem cells. In certain embodiments, the tumors harborcancer stem cells and their associated markers as set forth in Table 2above.

Suitable cancer stem cell antigens may be identified: (i) throughpublicly available information, such as published and unpublishedexpression profiles including cell surface antigens of cancer stem cellsof a particular tumor type or adult stem cells for a particular tissuetype (e.g. Table 2), and/or (ii) by cloning cancer stem cells or adultstem cells of a particular tumor or tissue type, respectively, in orderto determine their expression profiles and complement of cell surfaceantigens. Cloning of normal stem cells is a technique routinely employedin the art (Uchida et al., “Heterogeneity of hematopoeitic stem cells,”Curr. Opin. Immunol. 5:177-184 (1993)). In fact, this same technique isused to identify normal stem cells and cancer stem cells. Moreover,assumption that a proportion of normal stem cell gene products, e.g.cell surface antigens, will also be present on cancer stem cells derivedfrom the same tissue type has proven an effective way to identify cancerstem cell gene products and cancer stem cells. For example, knowledgethat the normal hematopoietic stem cell was CD34+/CD38− resulted in thedetermination that acute myeloid leukemia (AML) stem cells is similarlyCD34+/CD38−. This indeed was confirmed by standard stem cell cloningtechniques (See Bonnet et al., “Human acute myeloid leukemia isorganized as a hierarchy that originates from a primitive hematopoieticcell,” Nat. Med. 3:730-737 (1997)). Brain cancer stem cells weresimilarly isolated using a marker of normal (brain) stem cells, in thiscase CD133 (See Singh et al. “Identification of human brain tumorinitiating cells,” Nature 432(7015):396-401 (2004)).

In certain embodiments using flow cytometry of a sample, the Hoechst dyeprotocol can be used to identify cancer stem cells in tumors. Briefly,two Hoechst dyes of different colors (typically red and blue) areincubated with tumor cells. The cancer stem cells, in comparison withbulk cancer cells, over-express dye efflux pumps on their surface thatallow these cells to pump the dye back out of the cell. Bulk tumor cellslargely have fewer of these pumps, and are therefore relatively positivefor the dye, which can be detected by flow cytometry. Typically agradient of dye positive (“dye⁺”) vs. dye negative (“dye⁻”) cellsemerges when the entire population of cells is observed. Cancer stemcells are contained in the dye⁻ or dye low (dye^(low)) population. Foran example of the use of the Hoechst dye protocol to characterize a stemcell population see Goodell et al., “A leukemic stem cell with intrinsicdrug efflux pump capacity in acute myeloid leukemia,” Blood,98(4):1166-1173 (2001) and Kondo et al., “Persistence of a smallpopulation of cancer stem-like cells in the C6 glioma cell line,” Proc.Natl. Acad. Sci. U.S.A. 101:781-786 (2004). In this way, flow cytometrycould be used to measure cancer stem cell amount pre- and post-therapyto assess the change in cancer stem cell amount arising from a giventherapy or regimen.

In other embodiments using flow cytometry of a sample, the cells in thesample may be treated with a substrate for aldehyde dehydrogenase thatbecomes fluorescent when catalyzed by this enzyme. For instance, thesample can be treated with BODIPY®-aminoacetaldehyde which iscommercially available from StemCell Technologies Inc. as Aldefluor®.Cancer stem cells express high levels of aldehyde dehydrogenase relativeto bulk cancer cells and therefore become brightly fluorescent uponreaction with the substrate. The cancer stem cells, which becomefluorescent in this type of experiment, can then be detected and countedusing a standard flow cytometer. In this way, flow cytometry could beused to measure cancer stem cell amount pre- and post-therapy to assessthe change in cancer stem cell amount arising from a given therapy orregimen.

In other embodiments, a sample (e.g., a tumor or normal tissue sample,blood sample or bone marrow sample) obtained from the patient iscultured in in vitro systems to assess the cancer stem cell population.For example, tumor samples can be cultured on soft agar, and the amountof cancer stem cells can be correlated to the ability of the sample togenerate colonies of cells that can be visually counted. Colonyformation is considered a surrogate measure of stem cell content, andthus, can be used to quantitate the amount of cancer stem cells. Forinstance, with hematological cancers, colony-forming assays includecolony forming cell (CFC) assays, long-term culture initiating cell(LTC-IC) assays, and suspension culture initiating cell (SC-IC) assays.In this way, the colony-forming or related a assay, such as long-termperpetuation/passage of a cell line, could be used to measure cancerstem cell amount pre- and post-therapy to assess the change in cancerstem cell amount arising from a given therapy or regimen.

In other embodiments, sphere formation is measured to determine theamount of cancer stem cells in a sample (e.g., cancer stem cells formthree-dimensional clusters of cells, called spheres) in appropriatemedia that is conducive to forming spheres. Spheres can be quantitatedto provide a measure of cancer stem cells. See Singh et al.,“Identification of a Cancer Stem Cell from Human Brain Tumors,” CancerRes. 63: 5821-5828 (2003). Secondary spheres can also be measured.Secondary spheres are generated when the spheres that form from thepatient sample are broken apart, and then allowed to reform. In thisway, the sphere-forming assay could be used to measure cancer stem cellamount pre- and post-therapy to assess the change in cancer stem cellamount arising from a given therapy or regimen.

In other embodiments, the amount of cancer stem cells in a sample can bedetermined with a cobblestone assay. Cancer stem cells from certainhematological cancers form “cobblestone areas” (CAs) when added to aculture containing a monolayer of bone marrow stromal cells. Forinstance, the amount of cancer stem cells from a leukemia sample can beassessed by this technique. The tumor samples are added to the monolayerof bone marrow stromal cells. The leukemia cancer stem cells, more sothan the bulk leukemia cells, have the ability to migrate under thestromal layer and seed the formation of a colony of cells which can beseen visually under phase contrast microscopy in approximately 10-14days as CAs. The number of CAs in the culture is a reflection of theleukemia cancer stem cell content of the tumor sample, and is considereda surrogate measure of the amount of stem cells capable of engraftingthe bone marrow of immunodeficient mice. This assay can also be modifiedso that the CAs can be quantitated using biochemical labels ofproliferating cells instead of manual counting, in order to increase thethroughput of the assay. See Chung et al., “Enforced expression of anFlt3 internal tandem duplication in human CD34+ cells confers propertiesof self-renewal and enhanced erythropoiesis,” Blood 105(1):77-84 (2005).In this way, the cobblestone assay could be used to measure cancer stemcell amount pre- and post-therapy to assess the change in cancer stemcell amount arising from a given therapy or regimen.

In other embodiments, a sample (e.g., a tumor or normal tissue sample,blood sample or bone marrow sample) obtained from the patient isanalyzed in in vivo systems to determine the cancer stem cellpopulation. In certain embodiments, for example, in vivo engraftment isused to quantitate the amount of cancer stem cells in a sample. In vivoengraftment involves implantation of a human specimen with the readoutbeing the formation of tumors in an animal such as in immunocompromisedor immunodeficient mice (such as NOD/SCID mice). Typically, the patientsample is cultured or manipulated in vitro and then injected into themice. In these assays, mice can be injected with a decreasing amount ofcells from patient samples, and the frequency of tumor formation can beplotted vs. the amount of cells injected to determine the amount ofcancer stem cells in the sample. Alternatively, the rate of growth ofthe resulting tumor can be measured, with larger or more rapidlyadvancing tumors indicating a higher cancer stem cell amount in thepatient sample. In this way, an in vivo engraftment model/assay could beused to measure cancer stem cell amount pre- and post-therapy to assessthe change in cancer stem cell amount arising from a given therapy orregimen.

The amount of cancer stem cells in a specimen can be compared to apredetermined reference range and/or an earlier amount of cancer stemcells previously determined for the subject (either prior to, or duringtherapy) in order to gauge the subject's response to the treatmentregimens described herein. In a specific embodiment, a stabilization orreduction in the amount of cancer stem cells relative to a predeterminedreference range and/or earlier cancer stem cell amount previouslydetermined for the subject (either prior to, or during therapy)indicates that the therapy or regimen was effective and thus possibly animprovement in the subject's prognosis, whereas an increase relative tothe predetermined reference range and/or cancer stem cell amountdetected at an earlier time point indicates that the therapy or regimenwas ineffective and thus possibly the same or a worsening in thesubject's prognosis. The cancer stem cell amount can be used with otherstandard measures of cancer to assess the prognosis of the subjectand/or efficacy of the therapy or regimen: such as response rate,durability of response, relapse-free survival, disease-free survival,progression-free survival, and overall survival. In certain embodiments,the dosage, frequency and/or duration of administration of a therapy ismodified as a result of the determination of the amount of cancer stemcells at various time points which may include prior to, during, and/orfollowing therapy.

The present invention also relates to methods for determining that acancer therapy or regimen is effective at targeting and/or impairingcancer stem cells by virtue of monitoring cancer stem cells over timeand detecting a stabilization or decrease in the amount of cancer stemcells during and/or following the course of the cancer therapy orregimen.

In a certain embodiment, a therapy or regimen may be described ormarketed as an anti-cancer stem cell therapy or regimen based on thedetermination that a therapy or regimen is effective at targeting and/orimpairing cancer stem cells by virtue of having monitored or detected astabilization or decrease in the amount of cancer stem cells duringtherapy.

5.5 Methods of Monitoring Cancer Cells

As part of the prophylactically and/or therapeutically effectiveregimens of the invention, the amount of cancer cells (alone or incombination with the amount of cancer stem cells) can bemonitored/assessed using standard techniques known to one of skill inthe art. In certain embodiments of the prophylactically and/ortherapeutically effective regimens of the invention, the regimens resultin a stabilization or reduction in the amount (expressed, e.g., as apercentage) of cancer cells in the subject. In one embodiment, thesubject undergoing the regimen is monitored to determine whether theregimen has resulted in a stabilization or reduction in the amount(expressed, e.g., as a percentage) of cancer cells in the subject.

In some embodiments, the amount of cancer cells is assessed in a subjectusing techniques described herein or known to one of skill in the art.In other embodiments, the amount of cancer cells is detected in asample. Such samples include, but are not limited to, biological samplesand samples derived from a biological sample. In certain embodiments, inaddition to the biological sample itself or in addition to materialderived from the biological sample such as cells, the sample used in themethods of this invention comprises added water, salts, glycerin,glucose, an antimicrobial agent, paraffin, a chemical stabilizing agent,heparin, an anticoagulant, or a buffering agent. In certain embodiments,the biological sample is blood, serum, urine, bone marrow, orinterstitial fluid. In another embodiment, the sample is a tissuesample. In a particular embodiment, the tissue sample is breast, colon,lung, liver, ovarian, pancreatic, prostate, renal, bone, or skin tissue.In a specific embodiment, the tissue sample is a biopsy, including atumor biopsy. The amount of biological sample taken from the subjectwill vary according to the type of biological sample and the method ofdetection to be employed. In a particular embodiment, the biologicalsample is blood, serum, or urine and the amount of blood, serum, orurine taken from the subject is 0.1 ml, 0.5 ml, 1 ml, 5 ml, 10 ml ormore. In another embodiment, the biological sample is a tissue and theamount of tissue taken from the subject is less than 10 milligrams, lessthan 25 milligrams, less than 50 milligrams, less than 1 gram, less than5 grams, less than 10 grams, less than 50 grams, or less than 100 grams.

In accordance with the methods of the invention, a sample derived from abiological sample is one in which the biological sample has beensubjected to one or more pretreatment steps prior to the detectionand/or measurement of the cancer cell population in the sample. Incertain embodiments, a biological fluid is pretreated by centrifugation,filtration, precipitation, dialysis, or chromatography, or by acombination of such pretreatment steps. In other embodiments, a tissuesample is pretreated by freezing, chemical fixation, paraffin embedding,dehydration, permeablization, or homogenization followed bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In certain embodiments,the sample is pretreated by removing cells other than cancer cells fromthe sample, or removing debris from the sample prior to thedetermination of the amount of cancer cells in the sample according tothe methods of the invention.

The samples for use in the methods of this invention may be taken fromany animal subject, preferably mammal, most preferably a human. Thesubject from which a sample is obtained and utilized in accordance withthe methods of this invention includes, without limitation, anasymptomatic subject, a subject manifesting or exhibiting 1, 2, 3, 4, ormore symptoms of cancer, a subject clinically diagnosed as havingcancer, a subject predisposed to cancer, a subject suspected of havingcancer, a subject undergoing therapy for cancer, a subject that has beenmedically determined to be free of cancer (e.g., following therapy forthe cancer), a subject that is managing cancer, or a subject that hasnot been diagnosed with cancer.

In certain embodiments, the amount of cancer cells is assessed in asubject or a sample from a subject at least 1, 2, 4, 6, 7, 8, 10, 12,14, 15, 16, 18, 20, or 30, 60, 90 days 6 months, 9 months, 12 months,or >12 months after the subject begins receiving the regimen. In certainembodiments, the amount of cancer cells is assessed after a number ofdoses (e.g., after 1, 2, 5, 10, 20, 30, or more doses of a therapy). Inother embodiments, the amount of cancer cells is assessed after 2 weeks,1 month, 2 months, 1 year, 2 years, 3 years, 4 years, or more afterreceiving one or more therapies.

The amount of cancer cells in a sample can be expressed as thepercentage of, e.g., overall cells in the sample. In some embodiments,the sample is a blood sample or bone marrow sample, wherein the amountof cancer cells per unit of volume (e.g., 1 ml) or other measured unit(e.g., per unit field in the case of a histological analysis) isquantitated. The cancer cell population, in certain embodiments, can bedetermined as a percentage of the total blood cells.

In other embodiments, the sample from the patient is a tissue sample(e.g., a biopsy from a subject with or suspected or having canceroustissue), where the amount of cancer cells can be measured, for example,by immunohistochemistry or on the basis of the amount of cancer cellsper unit weight of the tissue.

The amount of cancer cells in the test sample can be compared with theamount of cancer cells measured in a reference sample(s) to assess theefficacy of the regimen. In one embodiment, the reference sample is asample from the subject undergoing therapy, at an earlier time point(e.g., prior to receiving the regimen as a baseline reference sample, orat an earlier time point while receiving the therapy). In thisembodiment, the therapy desirably results in a decrease in the amount ofcancer cells in the test sample as compared with the reference sample.In another embodiment, the reference sample is obtained from a healthysubject with no detectable cancer, or from a patient that is inremission for the same type of cancer. In this embodiment, the therapydesirably results in the test sample having an equal amount of cancercells as detected in the reference sample (e.g., no detectable cancercells).

If the reduction in the amount of cancer cells is judged too small, thenthe medical practitioner has a number of options to adjust the regimen.For instance, the medical practitioner can then either increase thedosage of the therapy administered, the frequency of the administration,the duration of administration, combine the therapy with anothertherapy(ies), halt the therapy, or any combination thereof.

The amount of cancer cells can be monitored/assessed using standardtechniques known to one of skill in the art. Cancer cells can bemonitored by, e.g., obtaining a sample, such as a tumor sample, bloodsample or bone marrow sample, from a subject and detecting cancer cellsin the sample. The amount of cancer cells in a sample (which may beexpressed as a percentage) can be assessed by detecting the expressionof antigens on cancer cells and/or by detecting the proliferation ofcancer cells. Techniques known to those skilled in the art can be usedfor measuring these activities. For example, cellular proliferation canbe assayed by 3H-thymidine incorporation assays and trypan blue cellcounts. Antigen expression can be assayed, for example, by immunoassaysincluding, but not limited to western blots, immunohistochemistry,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, flow cytometry,fluorescence-activated cell sorting (FACS) analysis, andimmunofluorescence.

The amount of cancer cells can be compared to a predetermined referencerange and/or an earlier amount of cancer cells determined for thesubject to gauge the subject's response to the regimens describedherein. In a specific embodiment, a reduction in the amount of cancercells relative to a predetermined reference range and/or earlier cancercell amount determined for the subject indicate an improvement in thesubject's prognosis or response to a therapy, whereas an increaserelative to the predetermined reference range and/or earlier cancer cellamount indicates the same or worse prognosis, or failure to respond to atherapy. In certain embodiments, the dosage, frequency and/or durationof administration of a therapy is modified as a result of the change inthe amount of cancer cells.

In some embodiments, the cancer cell population can bemonitored/assessed using gross measurements of the cancer cellpopulation. For example, in some embodiments, the cancer cell populationis determined using imaging methods such as computed tomography (CT),magnetic resonance imaging (MRI), ultrasound, X-ray imaging,mammography, radionuclide imaging, PET scan or bone scans.

In embodiments of the invention comprising treatment of solid tumors,the bulk size of the tumor may provide an estimate of the cancer cellpopulation. A number of known methods can be used to assess the bulksize of the tumor. Non-limiting examples of such methods include imagingmethods (e.g., computed tomography (CT), magnetic resonance imaging(MRI), PET scans, ultrasound, X-ray imaging, mammography, bone scans andradioisotope imaging), visual methods (e.g., colonoscopy, bronchoscopy,endoscopy), physical examination (e.g., prostate examination, breastexamination, lymph nodes examination, abdominal examination, generalpalpation), blood tests (e.g., prostate specific antigen (PSA) test,carcinoembryonic antigen (CEA) test, cancer antigen (CA)-125 test,alpha-fetoprotein (AFP)), bone marrow analyses (e.g., in cases ofhematological malignancies), histopathology, cytology and flowcytometry.

In some embodiments, the bulk tumor size can be measured by assessmentsbased on the size of tumor lesions determined from imaging methods. Inspecific embodiments, the assessments are performed in accordance withthe Response Evaluation Criteria In Solid Tumors (RECIST) Guidelines,which are set forth in Therasse, P. et al., “New Guidelines to Evaluatethe Response to Treatment in Solid Tumors,” J. of the Nat. Canc. Inst.92(3), 205-216 (2000). For instance, in specific embodiments, lesions inthe subject that are representative of bulk tumor size are selected sothat they are at least =20 mm in their longest diameter at baseline(prior to treatment) when conventional imaging techniques are used(e.g., conventional CT scan, MRI or X-ray) and lesions that are at least=10 mm in their longest diameter at baseline should be selected whenspiral CT scanning is used.

5.6 Methods of Monitoring Lymphocyte Cell Count, Neutrophil Cell Count,Platelet Count and Hemoglobin

As part of the prophylactically and/or therapeutically effectiveregimens of the invention, peripheral blood lymphocyte counts can bemonitered/assessed using standard techniques known to one of skill inthe art. Peripheral blood lymphocyte counts in a subject can bedetermined by, e.g., obtaining a sample of peripheral blood from saidsubject, separating the lymphocytes from other components of peripheralblood such as plasma using, e.g., Ficoll-Hypaque (Pharmacia) gradientcentrifugation, and counting the lymphocytes using trypan blue.Peripheral blood T-cell counts in a subject can be determined by, e.g.,separating the lymphocytes from other components of peripheral bloodsuch as plasma using, e.g., Ficoll-Hypaque (Pharmacia) gradientcentrifugation. Labeling the T-cells with an antibody directed to aT-cell antigen such as CD3, CD4, and CD8 which is conjugated to a FACSdetectable agent, such as FITC or phycoerythrin, and measuring theamount of T-cells by FACS. Further, the effect on a particular subset ofT cells (e.g., CD2+, CD4+, CD8+, CD45+, CD45RO+, CD45RA+, or CD8+RA+) orNK cells can be determined using standard techniques known to one ofskill in the art, such as FACS.

The subject's absolute neutrophil count (ANC) can be monitored/assessedusing standard techniques known to one of skill in the art. In someembodiments, the regimen includes monitoring the patient's ANC in orderto avoid the risk of the patient developing neutropenia.

The ANC can be calculated from measurements of the total number of whiteblood cells (WBC) and the numbers of neutrophils and bands (immatureneutrophils). The ANC can be determined manually by trained medicaltechnologists or by automated ANC results obtained from automatedhematology analyzers.

The subject's platelet count (PLT) can be monitored/assessed usingstandard techniques known to one of skill in the art. In someembodiments, the regimen includes monitoring the patient's plateletcount in order to avoid the risk of the patient developingthrombocytopenia or becoming blood transfusion dependent. Transfusionscan be given as determined by the physician.

The subject's hemoglobin (Hgb) can be monitored/assessed using standardtechniques known to one of skill in the art. In some embodiments, theregimen includes monitoring the patient's hemoglobin in order to avoidthe risk of the patient developing anemia or becoming transfusiondependent. Transfusions or growth factors (e.g. erythropoietin) can begiven as determined by the physician.

5.7 Biological Assays

5.7.1 In Vitro Assays

The compounds, pharmaceutical compositions and regimens of the inventioncan be tested in vitro and/or in vivo for their ability to reduce theamount of cancer cells and/or cancer stem cells, or inhibit theirproliferation. The ability of a compound or a regimen of the inventionto reduce the amount of cancer cells, cancer stem cells and/or immunecells (e.g., lymphocytes) or inhibit their proliferation can be assessedby: detecting the expression of antigens on cancer cells, cancer stemcells, and/or immune cells; detecting the proliferation or viability ofcancer cells, cancer stem cells and immune cells; detecting the effectorfunction of cancer cells and cancer stem cells. Techniques known tothose skilled in the art can be used for measuring these activities. Forexample, cellular proliferation can be assayed by ³H-thymidineincorporation assays and trypan blue cell counts. Antigen expression canbe assayed, for example, by immunoassays including, but not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, immunohistochemistry radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, and FACS analysis.

A compound, pharmaceutical composition, or regimen of the invention ispreferably tested in vitro and then in vivo for the desired therapeuticor prophylactic activity prior to use in humans. For example, assayswhich can be used to determine whether administration of a specificcompound is indicated include cell culture assays in which a patienttissue sample (e.g., a cancer cell or cancer stem cell) is grown inculture and exposed to, or otherwise contacted with, a compound of theinvention, and the effect of such compound upon the tissue sample isobserved. The tissue sample can be obtained by biopsy from the patient.This test allows the identification of the therapeutically mosteffective therapy (e.g., prophylactic or therapeutic agent) for eachindividual patient.

Determination of cell viability using the XTT assay: In some cases,CD34+ cells are isolated from human cord blood using magnetic beadscoated with anti-CD34 antibody. Isolated cells are then counted andaliquoted into 96-well plates and then incubated in the presence ofvarying concentrations of cantharidin or norcantharidin. Cell viabilityis measured by the addition of the XTT colorimetric reagent. Viabilityis determined by the absorbance of treated cultures at approximately450-500 nm compared to untreated cultures. In other cases, the cellsused in the assay may be a leukemia cell line, such as MV4; 11. Theassay can also be used to determine the time course of cell killing byvarious compounds by performing the XTT assay on cultures that areincubated with the compounds for varying periods of time.

Cobblestone assay: The cobblestone area-forming cell (CAFC) assayexploits a reproducible visual end point for the quantitation of cancerstem cells. Leukemia samples are added to adherent cultures of stromalcells, in some embodiments, MS-5 stromal cells. The cancer stem cells inthe culture will migrate below the MS-5 stromal cells and form a colonyof cells called a cobblestone that can be visually quantitated. To testthe effect of cantharidin or norcantharidin on the cancer stem cellpopulation using this assay, cells are first cultured in the presence ofthe drug. In some embodiments the cells are cultured for 16 hours. Afterthis incubation, the cells are added to the stromal cultures. Areduction in the cobblestone area formation in cultures that weretreated with the drug compared to the untreated cells represents cancerstem cell activity for the drug.

5.7.2 In Vivo Assays

The compounds, pharmaceutical compositions, and regimens of theinvention can be tested in suitable animal model systems prior to use inhumans. Such animal model systems include, but are not limited to, rats,mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animalsystem well-known in the art may be used. Several aspects of theprocedure may vary; said aspects include, but are not limited to, thetemporal regime of administering the therapeutic modalities (e.g.,prophylactic and/or therapeutic agents), whether such therapeuticmodalities are administered separately or as an admixture, and thefrequency of administration of the therapeutic modalities.

Animal models for cancer can be used to assess the efficacy of acompound or a combination therapy of the invention. Examples of animalmodels for lung cancer include, but are not limited to, lung canceranimal models described by Zhang & Roth (1994, In Vivo 8(5):755-69) anda transgenic mouse model with disrupted p53 function (see, e.g., Morriset al. J. La. State Med. Soc. 1998, 150(4):179-85). An example of ananimal model for breast cancer includes, but is not limited to, atransgenic mouse that overexpresses cyclin D1 (see, e.g., Hosokawa etal., Transgenic Res. 2001, 10(5), 471-8. An example of an animal modelfor colon cancer includes, but is not limited to, a TCR b and p53 doubleknockout mouse (see, e.g., Kado et al., Cancer Res. 2001, 61(6):2395-8).Examples of animal models for pancreatic cancer include, but are notlimited to, a metastatic model of PancO2 murine pancreaticadenocarcinoma (see, e.g., Wang et al., Int. J. Pancreatol. 2001,29(1):37-46) and nu-nu mice generated in subcutaneous pancreatic tumours(see, e.g., Ghaneh et al., Gene Ther. 2001, 8(3):199-208). Examples ofanimal models for non-Hodgkin's lymphoma include, but are not limitedto, a severe combined immunodeficiency (“SCID”) mouse (see, e.g., Bryantet al., Lab Invest. 2000, 80(4), 553-73) and an IgHmu-HOX11 transgenicmouse (see, e.g., Hough et al., Proc. Natl. Acad. Sci. U.S.A. 1998,95(23), 13853-8. An example of an animal model for esophageal cancerincludes, but is not limited to, a mouse transgenic for the humanpapillomavirus type 16 E7 oncogene (see, e.g., Herber et al., J. Virol.1996, 70(3):1873-81). Examples of animal models for colorectalcarcinomas include, but are not limited to, Apc mouse models (see, e.g.,Fodde & Smits, Trends Mol. Med. 2001, 7(8):369-73 and Kuraguchi et al.,Oncogene 2000, 19(50), 5755-63).

In certain in vivo techniques, an imaging agent, or diagnostic moiety,is used which binds to molecules on cancer cells or cancer stem cells,e.g., cancer cell or cancer stem cell surface antigens. For instance, afluorescent tag, radionuclide, heavy metal, or photon-emitter isattached to an antibody (including an antibody fragment) that binds to acancer stem cell surface antigen. Exemplary cancer stem cell surfaceantigens are listed above in Table 2. The medical practitioner caninfuse the labeled antibody into the patient either prior to, during, orfollowing treatment, and then the practitioner can place the patientinto a total body scanner/developer which can detect the attached label(e.g., fluorescent tag, radionuclide, heavy metal, photon-emitter). Thescanner/developer (e.g., CT, MRI, or other scanner, e.g. detector offluorescent label, that can detect the label) records the presence,amount/quantity, and bodily location of the bound antibody. In thismanner, the mapping and quantitation of tag (e.g. fluorescence,radioactivity, etc.) in patterns (i.e., different from patterns ofnormal stem cells within a tissue) within a tissue or tissues indicatesthe treatment efficacy within the patient's body when compared to areference control such as the same patient at an earlier time point or apatient who has no detectable cancer. For example, a large signal(relative to a reference range or a prior treatment date, or prior totreatment) at a particular location indicates the presence of cancerstem cells. If this signal is increased relative to a prior date itsuggests a worsening of the disease and failure of therapy or regimen.Alternatively, a signal decrease indicates that therapy or regimen hasbeen effective.

Similarly, in some embodiments of the invention, the efficacy of thetherapeutic regimen in reducing the amount of cancer cells in animals(including humans) undergoing treatment can be evaluated using in vivotechniques. In one embodiment, the medical practitioner performs theimaging technique with labeled molecule that specifically binds thesurface of a cancer cell, e.g., a cancer cell surface antigen. SeeSection 5.4, supra, lists certain cancer cell surface antigens. In thismanner, the mapping and quantitation of tag (e.g., fluorescence,radioactivity) in patterns within a tissue or tissues indicates thetreatment efficacy within the body of the patient undergoing treatment.

In a specific embodiment, the amount of cancer stem cells is detected invivo in a subject according to a method comprising the steps of: (a)administering to the subject an effective amount of a labeled cancerstem cell marker binding agent that specifically binds to a cell surfacemarker found on the cancer stem cells, and (b) detecting the labeledagent in the subject following a time interval sufficient to allow thelabeled agent to concentrate at sites in the subject where the cancerstem cell surface marker is expressed. In accordance with thisembodiment, the cancer stem cell surface marker-binding agent isadministered to the subject according to any suitable method in the art,for example, parenterally (e.g. intraveneously), or intraperitoneally.In accordance with this embodiment, the effective amount of the agent isthe amount which permits the detection of the agent in the subject. Thisamount will vary according to the particular subject, the label used,and the detection method employed. For example, it is understood in theart that the size of the subject and the imaging system used willdetermine the amount of labeled agent needed to detect the agent in asubject using imaging. In the case of a radiolabeled agent for a humansubject, the amount of labeled agent administered is measured in termsof radioactivity, for example from about 5 to 20 millicuries of ⁹⁹Tc.The time interval following the administration of the labeled agentwhich is sufficient to allow the labeled agent to concentrate at sitesin the subject where the cancer stem cell surface marker is expressedwill vary depending on several factors, for example, the type of labelused, the mode of administration, and the part of the subject's bodythat is imaged. In a particular embodiment, the time interval that issufficient is 6 to 48 hours, 6 to 24 hours, or 6 to 12 hours. In anotherembodiment the time interval is 5 to 20 days or 5 to 10 days. Thepresence of the labeled cancer stem cell surface marker-binding agentcan be detected in the subject using imaging means known in the art. Ingeneral, the imaging means employed depend upon the type of label used.Skilled artisans will be able to determine the appropriate means fordetecting a particular label. Methods and devices that may be usedinclude, but are not limited to, computed tomography (CT), whole bodyscan such as position emission tomography (PET), magnetic resonanceimaging (MRI), fluorescence, chemiluminescence, an imager which candetect and localize fluorescent label and sonography. In a specificembodiment, the cancer stem cell surface marker-binding agent is labeledwith a radioisotope and is detected in the patient using a radiationresponsive surgical instrument (Thurston et al., U.S. Pat. No.5,441,050). In another embodiment, the cancer stem cell surfacemarker-binding agent is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the cancer stem cell surfacemarker-binding agent is labeled with a positron emitting metal and isdetected in the patient using positron emission-tomography. In yetanother embodiment, the cancer stem cell surface marker-binding agent islabeled with a paramagnetic label and is detected in a patient usingmagnetic resonance imaging (MRI).

Any in vitro or in vivo (ex vivo) assays known to those skilled in theart that can detect and/or quantify cancer stem cells can be used tomonitor cancer stem cells in order to evaluate the prophylactic and/ortherapeutic utility of a cancer therapy or regimen disclosed herein forcancer or one or more symptoms thereof; or these assays can be used toassess the prognosis of a patient. The results of these assays then maybe used to possibly maintain or alter the cancer therapy or regimen.

5.7.3 Assessing Toxicity

The toxicity and/or efficacy of compounds, pharmaceutical compositions,and regimens of the invention can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Therapeutic regimens that exhibit large therapeutic indices arepreferred. While therapeutic regimens that exhibit toxic side effectsmay be used, care should be taken to design a delivery system thattargets such agents to the site of affected tissue in order to minimizepotential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the therapies for use inhumans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity to normal tissues. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any therapy used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof compounds in plasma may be measured, for example, by high performanceliquid chromatography.

5.8 Articles of Manufacture

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. Thepharmaceutical product may contain, for example, a conjugate of theinvention in a unit dosage form in a first container, and in a secondcontainer, sterile water for injection. Alternatively, the unit dosageform may be a solid suitable for oral, transdermal, intranasal, ortopical delivery.

In a specific embodiment, the unit dosage form is suitable forparenteral, intravenous, intramuscular, intranasal, oral,intraperitoneal, topical or subcutaneous delivery. Thus, the inventionencompasses solutions, preferably sterile, suitable for each deliveryroute.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician, or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, cancer cell counts, cancer stem cell counts, and othermonitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises a compound of the invention, and wherein said packagingmaterial includes instruction means which indicate that said compoundcan be used to prevent, manage, treat, and/or ameliorate one or moresymptoms associated with cancer, or one or more symptoms thereof byadministering specific doses and using specific dosing regimens asdescribed herein.

In a preferred embodiment, the article of manufacture includes labeledantibodies that bind to cancer cells, and preferably, that bind tocancer stem cells. As such, the article contains a method to monitor theefficacy of the therapeutic regimen, and to adjust, if need be, thetherapeutic dosages and/or regimens.

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with reagents for detecting,monitoring and/or measuring cancer stem cells. In one embodiment, thepharmaceutical pack or kit optionally comprises instructions for the useof the reagents provided for detecting and/or measuring cancer stemcells. In another embodiment, the pharmaceutical pack or kit optionallycomprises a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,for use or sale for human administration.

In an embodiment, the pharmaceutical pack or kit comprises in one ormore containers a cancer stem cell surface marker-binding agent. In aparticular embodiment, the agent is an antibody that selectively orspecifically binds to a cancer stem cell surface marker. In a particularembodiment, the agent is an antibody (including, e.g., human, humanized,chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)₂ fragments orepitope binding fragments), which cross-reacts with any cancer stem cellsurface marker. In another embodiment, the antibody cross reacts withany one of the cancer stem cell surface markers listed in Table 2. Inanother embodiment, the antibody reacts with any one of the cancer stemcell surface markers listed in Table 1 of U.S. Pat. No. 6,004,528 orTables 1, 2, or 3 of U.S. patent application Ser. No. 09/468,286, andU.S. Patent Application Publication Nos. 2006/0083682, 2007/0036800,2007/0036801, 2007/0036802, 2007/0041984, 2007/0036803, and2007/0036804, each of which is incorporated by reference herein. Inaccordance with this embodiment, the pharmaceutical pack or kitcomprises one or more antibodies which bind to cancer stem cell surfacemarkers, wherein each antibody binds to a different epitope of thecancer stem cell surface marker and/or binds to the cancer stem cellsurface marker with a different affinity.

For antibody based kits, the kit can comprise, for example: (1) a firstantibody (which may or may not be attached to a solid support) whichbinds to a cancer stem cell surface marker protein; and, optionally, (2)a second, different antibody which binds to either the cancer stem cellsurface marker protein bound by the first antibody, or the firstantibody and is conjugated to a detectable label (e.g., a fluorescentlabel, radioactive isotope or enzyme). The antibody-based kits may alsocomprise beads for conducting an immunoprecipitation. Each component ofthe antibody-based kits is generally in its own suitable container.Thus, these kits generally comprise distinct containers suitable foreach antibody. Further, the antibody-based kits may compriseinstructions for performing the assay and methods for interpreting andanalyzing the data resulting from the performance of the assay. As anexample, a kit may include an anti-CD34 antibody for positive selection,an anti-CD38 antibody for negative selection, and an anti-CD123 antibodyfor positive selection to isolate and/or quantify and/or assist in thedetermination of the amount of leukemia cancer stem cells (which areCD34+/CD38−/CD123+).

For nucleic acid micoarray kits, the kits generally comprise (but arenot limited to) probes specific for certain genes attached to a solidsupport surface. In other embodiments, the probes are soluble. In onesuch embodiment, probes can be either oligonucleotides or longer lengthprobes including probes ranging from 150 nucleotides in length to 800nucleotides in length. The probes may be labeled with a detectablelabel. The microarray kits may comprise instructions for performing theassay and methods for interpreting and analyzing the data resulting fromthe performance of the assay. The kits may also comprise hybridizationreagents and/or reagents necessary for detecting a signal produced whena probe hybridizes to a cancer stem cell surface marker nucleic acidsequence. Generally, the materials and reagents for the microarray kitsare in one or more containers. Each component of the kit is generally inits own a suitable container.

For Quantitative PCR, the kits generally comprise pre-selected primersspecific for certain cancer stem cell surface marker nucleic acidsequences. The Quantitative PCR kits may also comprise enzymes suitablefor amplifying nucleic acids (e.g., polymerases such as Taq), anddeoxynucleotides and buffers needed for the reaction mixture foramplification. The Quantitative PCR kits may also comprise probesspecific for the nucleic acid sequences associated with or indicative ofa condition. The probes may or may not be labeled with a fluorophore.The probes may or may not be labeled with a quencher molecule. In someembodiments, the Quantitative PCR kits also comprise components suitablefor reverse-transcribing RNA including enzymes (e.g. reversetranscriptases such as AMV, MMLV and the like) and primers for reversetranscription along with deoxynucleotides and buffers needed for thereverse transcription reaction. Each component of the quantitative PCRkit is generally in its own suitable container. Thus, these kitsgenerally comprise distinct containers suitable for each individualreagent, enzyme, primer and probe. Further, the quantitative PCR kitsmay comprise instructions for performing the assay and methods forinterpreting and analyzing the data resulting from the performance ofthe assay.

A kit can optionally further comprise a predetermined amount of anisolated cancer stem cell surface marker polypeptide or a nucleic acidencoding a cancer stem cell surface marker, e.g., for use as a standardor control. The diagnostic methods of the present invention can assistin conducting or monitoring a clinical study. In accordance with thepresent invention, suitable test samples, e.g., of serum or tissue,obtained from a subject can be used for diagnosis.

Based on the results obtained by use of the pharmaceutical pack or kit(i.e. whether the cancer stem cell amount has stabilized or decreased),the medical practitioner administering the cancer therapy or regimen maychoose to continue the therapy or regimen. Alternatively, based on theresult that the cancer stem cell amount has increased, the medicalpractitioner may choose to continue, alter or halt the therapy orregimen.

6. EXAMPLES

The following examples are illustrative, and should not be viewed aslimiting the scope of the present invention. Reasonable variations, suchas those that occur to a reasonable artisan, can be made herein withoutdeparting from the scope of the present invention.

6.1 Example 1

6.1.1 Patients and Study Design

The following example describes the results of a clinical study in whicha diphtheria toxin-interleukin-3 conjugate was administered to patientssuffering from acute myeloid leukemia (AML).

Patients were diagnosed with AML based on bone marrow biopsy and eitherrelapsed disease or poor-risk AML (treatment-related, priormyelodysplastic syndrome (MDS), patient age >70 years, or unfavorablecytogenetics and not candidate for allogeneic transplantation). Patientshad to have a performance status <2, WBC <10,000/L, bilirubin <1.5mg/dL, transaminases <2.5× upper limit normal, albumin >3 g/dL,creatinine <1.5 mg/dL, adequate cardiac reserve (EF>40%), anti-DTpretreatment serum concentration <2.4 μg/ml, be willing to give informedconsent and be treated at an approved site, be willing to use anapproved form of birth control while on study, have no concurrentserious medical problems or uncontrolled infections or DIC or pregnancy,not have active CNS leukemia, not have had a myocardial infarctionwithin the past six months, not require oxygen, and not have an allergyto diphtheria toxin.

Patients were admitted to the hospital, given allopurinol, normalsaline, moxifloxacin, fluconazole, vitamin K, acetaminophen,diphenhydramine, and hydrocortisone and inter-patient escalating dosesof DT388IL-3 IV over 15 minutes on M-W-F for two weeks. Cohorts of atleast 3 patients at each dose level were treated. Patients weremonitored for toxicities using the NCI Common Terminology Criteria forAdverse Events (CTCAE) version 3.0. Vital signs were measured frequentlyon the days of treatment. Careful input/output was recorded daily. CBC,CMP, coagulation panel, LDH, uric acid, and magnesium were tested daily.Blood was drawn for clinical pharmacology studies includingpharmacokinetics and immune response. Bone marrow biopsies were repeatedon day 15, 30, 60, and every three months until relapse. Responses weremeasured based on the revised recommendations of the internationalworking group.

6.1.2 Results

Forty-nine AML patients have been screened and twenty-seven patientstreated (Table 3). The median age of treated patients was 59 years(range, 25-81 years). There were thirteen males and fourteen females.Disease was de novo in two, first relapse in ten, second relapse inseven, and refractory in eight patients. Three patients had a history ofMDS, and one had a history of secondary AML. One patient each hadpreviously received an autologous or allogeneic stem cell transplant.Cytogenetics were unfavorable in ten, intermediate in sixteen, and notdone in one. Seven patients were treated with 4 μg/kg, eight patientswere treated with 5.3 μg/kg, eleven patients treated with 7.1 μg/kg, andone patient treated with 9 μg/kg DT₃₈₈IL-3 (Table 4). Drug-relatedtoxicities were mild to moderate and transient including fever, chills,hypotension, hypoxemia, and hypoalbuminemia. Among twenty-sevenevaluable patients, we have observed one on-going CR of 6+ monthsduration, two partial remissions (PRs) lasting one and two+ months andthree minimal responses with clearance of peripheral blasts and marrowblast cytoreductions of 89%, 90% and 93% lasting one to two months(Table 5 and FIG. 4).

Toxicities to date on the clinical study have been mild to moderate.Fevers occurred but responded to acetaminophen and cooling methods.Hypotension and transient uremia responded to hydration. Hypoxemia andhypoalbuminemia reversed with albumin infusions and diuresis. Nosignificant liver dysfunction has been seen. After the clinical study ofDT₃₈₈GMCSF, we established a preclinical model for the liver toxicityusing DT fusion molecules with murine GMCSF and IL-3, DT₃₈₈mGMCSF andDT₃₈₈mIL-3, respectively. 27 Rats treated with DT₃₈₈mGMCSF but notDT₃₈₈mIL-3 showed Kupffer cell injury, hepatocyte swelling andtransaminasemia. The lack of IL-3 receptor on Kupffer cells appears toprotect from liver damage. Monkey and clinical studies to date confirmsthat finding.

The above results clearly show that the diphtheria toxin-interleukin-3conjugate was selectively cytotoxic to leukemic cells relative to normalhematopoietic cells and produced clinical remissions in human patients.

TABLE 3 Results of DT₃₈₈IL3 treated patients-toxicities, immuneresponse, and clinical response* Anti-DT antibody Patient Dose levelToxicities (μg/mL) Clinical No. (ug/kg) Gr2 CTCV3 d1 d15 d30 Response 14 N-V, Trans 0.8 23 235 0 2 4 0 2.5 ND ND 0 3 4 F, N-V, Alb, Hypo 0 ND36 0 4 4 F, Alb 0 ND ND 0 5 4 F, Alb 0 ND 48 0 6 4 Hypo 0.9 ND 36 0 7 4Alb 2.2 1 6.2 0 8 5.32 Alb 1 221 263 0 9 5.32 Alb, Trans 0.8 440 ND PR10 5.32 Alb 0.5 ND 1.1 0 11 5.32 F, Alb, Trans 2.5 ND ND 0 12 5.32 Hypo,F, Alb 1.3 600 ND 0 13 5.32 Alb 1.5 ND ND MR 14 5.32 Alb, Trans 0.3 0.3ND MR 15 5.32 Alb, Trans 0 1.6 29 MR 16 7.07 Alb, Trans 1 0 ND 0 17 7.07Alb 1.2 ND ND 0 18 7.07 F, Alb, Dysp 0.7 0.4 ND 0 19 7.07 VLS, Alb, Dysp0.8 8.3 22.4 CR 20 7.07 Alb, Trans 0.4 ND ND 0 21 7.07 Alb, Trans 2.11.5 4.2 0 22 7.07 Alb, Trans 1.7 1.2 ND 0 23 7.07 F, Alb, VLS 2.2 32 104PR 24 7.07 0 4.3 ND 4 0 25 7.07 Alb 3.8 ND ND 0 26 7.07 F, alb, Trans0.5 ND ND 0 27 9.4 F, Alb, Trans 3 ND ND 0 28 9.4 F, Alb 1.3 300 ND 0 297.07 F, Alb 1.5 ND ND 0 30 9.4 Alb 3 0.8 ND 0 31 9.4 F, Trans 2.3 ND ND0 32 9.4 Alb, Trans 2.2 3.1 ND 0 33 9.4 Alb, Trans 1.2 ND ND 0 34 9.4Alb, Trans 2.2 ND 306 0 35 9.4 Alb 0.8 252 ND 0 36 12.5 Alb, Trans 1.311.2 16.8 PR *F = fever, N-V = nausea and vomiting, Trans =transaminasemia, VLS = vascular leak syndrome, Alb = hypoalbuminemia,Hypo = hypotension, Dysp = dyspnea, ND = not determined, MR = minimalresponse, PR = partial response, CR = complete response.

TABLE 4 Dose level and drug-related toxic effects of DT₃₈₈IL3-treatedAML patients Patient Dose no. μg/kg/day Drug-related Gr 2 or higher sideeffects (CTC toxicity grade) 1 4 Gr 2 nausea; Gr 2 vomiting; Gr 2 ALT 24 None 3 4 Gr 2 hypotension; Gr 2 sinus tachycardia; Gr 2 fever; Gr 2weight gain; Gr 2 nausea; Gr 2 vomiting; Gr 2 hypoalbuminemia; 4 4 Gr 2fever; Gr 2 hypoalbuminemia; Gr 2 hypocalcemia 5 4 Gr 2 fever; Gr 2rigors/chills; Gr 2 CPK; Gr 2 hypoalbuminemia; Gr 2 hypocalcemia 6 4 Gr2 hypotension; Gr 2 hypocalcemia 7 4 Gr 2 hypoalbuminemia 8 5.32 Gr 2hypoalbuminemia 9 5.32 Gr 2 hypoalbuminemia; Gr 2 hypocalcemia; Gr 2AST; Gr 2 ALT 10 5.32 Gr 2 supraventicular tachycardia; Gr 2hypoalbuminemia 11 5.32 Gr 2 fever; Gr 2 hypoalbuminemia; Gr 2hypocalcemia; Gr 2 AST; Gr 2 ALT 12 5.32 Gr 2 hypotension; Gr 2 fever;Gr 2 rigors/chills; Gr 2 weight gain; Gr 2 hypoalbuminemia; Gr 2hypocalcemia 13 5.32 Gr 2 hypoalbuminemia 14 5.32 Gr 2 hypoalbuminemia;Gr 2 hypocalcemia; Gr 2 ALT 15 5.32 Gr 2 fatigue; Gr 2 rigors/chills; Gr2 hypoalbuminemia; Gr 2 hypocalcemia; Gr 2 ALT 16 7.07 Gr 2hypoalbuminemia; Gr 2 hypocalcemia; Gr 2 ALT 17 7.07 Gr 2hypoalbuminemia 18 7.07 Gr 2 hypertension; Gr 2 fatigue; Gr 2 fever; Gr2 rash/desquamation; Gr 2 hypoalbuminemia; Gr 2 muscle weakness, wholebody/generalized; Gr 2 dyspnea 19 7.07 Gr 2 acute vascular leaksyndrome; Gr 2 hypertension; Gr 2 hypoalbuminemia; Gr 2 hypocalcemia; Gr2 dyspnea; Gr 2 hypoxia 20 7.07 Gr 2 hyperbilirubinemia; Gr 2hyperglycemia; Gr 2 hypoalbuminemia; Gr 2 hypocalcemia; Gr 2 AST; Gr 2ALT 21 7.07 Gr 2 hypoalbuminemia, Gr 2 AST, Gr 2 ALT 22 7.07 None 237.07 Gr 2 fever; Gr 2 hypocalcemia; Gr 2 hypoalbuminemia; Gr 2 acutevascular leak syndrome 24 7.07 None 25 7.07 Gr 2 hypoalbuminemia 26 7.07Gr 2 AST 27 9 Gr 2 fever; Gr 2 ALT; Gr 2 hypoalbuminemia

TABLE 5 DT₃₈₈IL3 Clinical Responses Length of Patient Pre-treatmentOverall response no. blast % Response (mos) 9 50% PR 1 13 69% MR with93% 2 reduction* 14 90% MR with 89% 1 reduction* 15 80% MR with 90% 1reduction* 19 30% CR ongoing for >6 23 39% PR ongoing for >2*Cytoreduction calculated from change in marrow blast index = % blasts ×% cellularity.

6.2 Example 2

6.2.1 Patients and Methods

Patients had to have AML based on bone marrow biopsy and either disease,relapsed disease, refractory disease or poor-risk AML(treatment-related, prior MDS, patient age >70 years, or unfavorablecytogenetics and not candidate for allogeneic transplantation). Patientshad to have a performance status <2, WBC <10,000/ml, bilirubin <1.5mg/dL, transaminases <2.5× upper limit of normal, albumin >3 g/dL,creatinine <1.5 mg/dL, adequate cardiac reserve (EF>40%), anti-DTpretreatment serum concentration <2.4 mg/ml, be willing to give informedconsent and be treated at an approved site, be willing to use anapproved form of birth control while on study, not have concurrentserious medical problems or uncontrolled infections or DIC or pregnancy,not have active CNS leukemia, not have had a myocardial infarctionwithin the past six months, not require oxygen, and not have an allergyto DT.

Patients received 15 minute infusions of DT₃₈₈IL-3 three times weeklyfor two weeks with interpatient dose escalation at doses of 4-12.5m/kg/dose.

6.2.2 Results—Patient Characteristics

Seventy-five AML patients have been screened to date and thirty-sixpatients treated (Table 6). The median age of treated patients was 60years (range, 25-81 years). There were twenty males and sixteen females.Disease was de novo AML in four, first relapse AML in eleven, secondrelapse AML in eight, and refractory AML in twelve patients. One patienthad MDS. Seven AML patients had a history of MDS, and one had a historyof secondary AML. One patient each had previously received an autologousor allogeneic stem cell transplant. Cytogenetics were unfavorable intwelve including the MDS patient, intermediate in twenty-one, and notdone in three. Seven patients were treated with 4 mg/kg, eight patientswere treated with 5.3 m/kg, twelve patients treated with 7.1 mg/kg, andeight patients treated with 9.4 μg/kg, one patient treated with 12.5μg/kg DT₃₈₈IL-3.

TABLE 6 Clinical characteristics of DT₃₈₈IL-3-treated AML patients AgePatient (yrs)/ Disease no. Sex Status Treatment History Cytogenetics  138/M 1^(st) rel 7 + 3; HIDAC Normal  2 53/F 2^(nd) rel 7 + 3/Ida/Ara-C;7 + 3/Ida/Ara-C; 5 + 3/Ida/Ara-c; Mylotarg +8  3^(b) 67/F 1^(st) relCarbo/Taxol; Gleevec then Ida/Cytarabine; Mylotarg −4, −16, −19, del 5,+7  4^(a) 67/F 2^(nd) rel Ida/Ara-C; Mylotarg; Campath/Cytoxan; Allostem; Normal Mylotarg; DLI; DLI; Mylotarg; DLI  5 57/M 1^(st) rel7&3/Cytarabine/Dauno; 5&2/Cytarabine/dauno; Cytarabine/Mylotarg; +13Cytoxan/VP-16  6^(a) 54/M 2^(nd) rel Ara-C/Ida; Ara-C/Gem/CPT-11;Ara-C/Etoposide; −7 Stem cell transplant; Busulphace/VP-16; Autologousstem  7^(b) 51/M 2^(nd) rel 7 + 3 + 3; Ara-C/L-asparaginase;Ara-C/VP-16, Busulfari/VP-16 plus auto t (6; 12) stem;Ara-C/Mito/L-asparaginase; Ara-C/Mito/L-asparaginase; Mylotarg  8 62/M1^(st) rel Cytarabine/Dauno t(3; 21) (q26; q26)  9 63/M 1^(st) relCytarabine/Dauno Normal 10 69/M 1^(st) rel Dauno/Ara-C; Dauno/Ara-C;Dauno/Ara-C; Dauno/Ara-C Normal 11 54/F Ref ERYC/Ida (7 + 3); ERYCNormal 12 81/F De Novo +8, +9 13 76/M 2^(nd) rel Ida/Ara-C;VP16/Mito/Ara-C; Mylotarg; Vion/Timidor; Gem/Fludaraine/Mito Normal 1467/F Ref ERYC/Ida (7 + 3) induction t(11; 18) (q25; q21), del (9)(p22p24); +8 15 25/F 2^(nd) rel 7 + 3 induction therapy; miniVP-16/Cytoxan/Ara-C; CECA re-induction t(9; 11) (p22; q23) followed by7 + 3 16 44/F Ref HIDAC/dauno; VP-16/Cyclophosphamide Normal 17 62/M Ref7 + 3 induction therapy; 5 + 2/Cytarabine/Asparaginase;Cloretazine/Temodar; t(1; 4) (q42; q21); t(4; 12) (q12; p13)Mito/Gem/Fludarabine del (7) (q22) 18 62/F 1^(st) rel 7 + 3 inductionNormal 19 72/F Ref 7 + 3 induction Normal 20 62/M 1^(st) relCytarabine/Ida add (2) (p21), acid (3) (p25), −4, −7, sl, del (17)(q23), sdll, del (11) (q23) 21^(b) 59/F 1^(st) rel Ara-C/dauno;VP-16/cyclo salvage t (1; 5) 22 32/M Ref Ara-C/dauno; VP-16/Mito;Ara-C/dauno; VP-16/Cytoxan del (7) (q22q34) 23 73/F 1^(st) relCyclosporin, daunorubicin, cytarabine +11, −12, der (17) t (12; 17)(q10; p12) 24 33/M 2^(nd) rel ADE-10; MACE/Midac; Hydrea/leukophoresis;Cytosine/Ara-C Normal 25^(b) 66/F Ref Revlimid; 7 + 3,L-001281814/MK0457 del (5) (q23), −12, −13, add (16) (q22), +mar 20 2673/F Ref Ara-C/dauno; 7 + 3 Normal 27 70/F De Novo ND 28 60/M Ref 7 + 3del (5) 29 41/F 2^(nd) rel 7 + 3; Hi-Dos Cytarabine; 7 + 3 re-inductionNormal 30 32/M Ref HIDAC/dauno; VP-16/Cyclophosphamide; HIDAC ND 31^(b)77/M De Novo Normal 32^(b) 72/M Ref Ara-C −Y 33 79/M 1^(st) rel 7 + 3;Hi-Dos Cytarabine Normal 34 77/M De Novo ND 35^(b) 68/M Ref 7 + 3, Highdose Ara-C, PT-523 (q5), del (7), t (16; 17) 36 71/M MDS 5-azacitidine;decitabine −7 ^(a)Patient 4 had a allogeneic transplant, and patient 6had a autologous transplant. ^(b)Patient 3 had history of secondary AML.Patient 7, 21, 25, 31, 32, and 35 had a prior history of MDS.

6.2.3 Results—Toxicities

Drug-related toxicities were mild to moderate and transient includingfever, chills, hypotension, vascular leak syndrome, hypoxemia,hypocalcemia, transaminasemia and hypoalbuminemia (Table 7 and FIG. 5).There is no correlation of dose level with toxicity incidence or grade.

TABLE 7 Results of DT388IL3 treated patients-toxicities,pharmacokinetics, immune response, and clinical response* Cmax Anti-DTantibody Patient Dose level Toxicities d1/d12 (mg/mL) Clinical No.(ug/kg) Gr2 CTCV3 (mg/mL) d1 d15 d30 Response 1 4 N-V, Trans 0 0 0.8 23235 0 2 4 0 0 0 2.5 ND ND 0 3 4 F, N-V, Alb, Hypo 0 0.18 0 ND 36 0 4 4F, Alb ND ND 0 ND ND 0 5 4 F, Alb ND ND 0 ND 48 0 6 4 Hypo ND ND 0.9 ND36 0 7 4 Alb 0 0 2.2 1 6.2 0 8 5.32 Alb 0.19 0 1 221 263 0 9 5.32 Alb,Trans 0.14 0.15 0.8 440 ND PR 10 5.32 Alb 0.22 ND 0.5 ND 1.1 0 11 5.32F, Alb, Trans 0 ND 2.5 ND ND 0 12 5.32 Hypo, F, Alb 0.34 0 1.3 600 ND 013 5.32 Alb 0 0.3 1.5 ND ND MR 14 5.32 Alb, Trans 0.38 0.36 0.3 0.3 NDMR 15 5.32 Alb, Trans 0.06 0.29 0 1.6 29 MR 16 7.07 Alb, Trans ND 0.21 10 ND 0 17 7.07 Alb ND ND 1.2 ND ND 0 18 7.07 F, Alb, Dysp 0.22 0.37 0.70.4 ND 0 19 7.07 VLS, Alb, Dysp 0.32 0.54 0.8 8.3 22.4 CR 20 7.07 Alb,Trans 0.48 ND 0.4 ND ND 0 21 7.07 Alb, Trans 0.08 0.35 2.1 1.5 4.2 0 227.07 Alb, Trans 0.13 0.29 1.7 1.2 ND 0 23 7.07 F, Alb, VLS 0.61 ND 2.232 104 PR 24 7.07 0 0 0.38 4.3 ND 4 0 25 7.07 Alb 0.11 ND 3.8 ND ND 0 267.07 F, alb, Trans 0.23 ND 0.5 ND ND 0 27 9.4 F, Alb, Trans 0.18 ND 3 NDND 0 28 9.4 F, Alb 0.27 ND 1.3 300 ND 0 29 7.07 F, Alb ND ND 1.5 ND ND 030 9.4 Alb 0.15 0.32 3 0.8 ND 0 31 9.4 F, Trans 0.23 ND 2.3 ND ND 0 329.4 Alb, Trans 0.26 0.38 2.2 3.1 ND 0 33 9.4 Alb, Trans 0.37 ND 1.2 NDND 0 34 9.4 Alb, Trans 0.55 ND 2.2 ND 306 0 35 9.4 Alb 0.23 0 0.8 252 ND0 36 12.5 Alb, Trans 0.34 ND 1.3 11.2 16.8 PR *F = fever, N-V = nauseaand vomiting, Trans = transaminasemia, VLS = vascular leak syndrome, Alb= hypoalbuminemia, Hypo = hypotension, Dysp = dyspnea, ND = notdetermined, MR = minimal response, PR = partial response, CR = completeresponse.

6.2.4 Results—Immune Response

Pretreatment antibody titers ranged from 0 to 4.3 μg/ml (mean=2.3μg/ml); day 15 antibody titers were 0 to 600 μg/ml (mean=92 μg/ml); day30 antibody titers were 1.1 to 306 μg/ml (mean=81 μg/ml). Based on highantibody titers of >8 m/ml, all 25 samples were low pretreatment; ninesamples were low at day 15 and nine samples high at day 15; four sampleswere low at day 30 and ten samples high at day 30. Cmax did notcorrelate with response (p=0.23).

6.2.5 Results—Clinical Response

Among thirty-six evaluable patients, the following were observed: onecytogenetic AML CR for 8 months; two AML partial remissions (PRs)lasting one and three months; three AML minimal responses with clearanceof peripheral blasts and marrow blast cytoreductions of 89%, 90% and 93%lasting one to two months; and one MDS partial remission lasting greaterthan one month with reduction of blasts from 10% to 2% and normalizationof peripheral counts (Table 8 and FIG. 8A-D).

TABLE 8 DT₃₈₈IL3 Clinical Responses Length of Patient Dose Pre-treatmentresponse no. Level blast % Overall Response (mos) 9 5.32 50% PR 1 135.32 69% MR with 93% 2 reduction* 14 5.32 90% MR with 89% 1 reduction*15 5.32 80% MR with 90% 1 reduction* 19 7.07 30% CR 8 23 7.07 39% PR 336 12.5 10% PR >1 *Cytoreduction calculated from change in marrow blastindex = % blasts × % cellularity.

7. EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings using no more thanroutine experimentation. Such modifications and equivalents are intendedto fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent, or patent application was specifically and individuallyindicated to be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

I claim:
 1. A method for inhibiting cancer cells in a human diagnosed with plasmacytoid dendritic cell cancer, comprising administering to said human an effective amount of a pharmaceutical composition comprising a human interleukin 3 (IL-3)-diphtheria toxin conjugate, wherein said conjugate is administered at a dose greater than 4 μg/kg to about 12.5 μg/kg, and wherein said method results in inhibition of the growth of plasmacytoid dendritic cell cancer cells.
 2. The method of claim 1, wherein the inhibition results in a reduction in the proliferation of plasmacytoid dendritic cell cancer cells, a stabilization in the amount of plasmacytoid dendritic cell cancer cells, a reduction in the amount of plasmacytoid dendritic cell cancer cells, a stabilization in the amount of plasmacytoid dendritic cell cancer blasts, a reduction in the amount of plasmacytoid dendritic cell cancer blasts, an improvement in hematopoietic function in the subject, and/or an improvement in the marrow blast index of the subject.
 3. The method of claim 2, wherein said stabilization or reduction is measured by blood tests; blast count; blast percentage; physical examination; complete blood count; flow cytometric analyses; bone marrow aspirate; bone marrow analyses; skin surveillance, hematopoietic function; marrow blast index; the amount of normal white blood cells, red blood cells, and/or platelets; histology; immunohistochemistry; frequency of transfusion; and/or bone marrow biopsy.
 4. The method of claim 1, wherein the cells of said plasmacytoid dendritic cell cancer express the IL-3 receptor.
 5. The method of claim 1, wherein the conjugate is administered at a dose of about 5.3 μg/kg, about 7.1 μg/kg, about 9.4 μg/kg, or about 12.5 μg/kg.
 6. The method of claim 1, wherein the conjugate is administered at a dose that is the maximum tolerated dose.
 7. The method of claim 1, wherein the conjugate is administered at least two times a week.
 8. The method of claim 1, wherein the conjugate is administered at least three times a week.
 9. The method of claim 1, wherein the conjugate is administered over a period of two weeks or more.
 10. The method of claim 1, wherein the conjugate is administered once every day for five days.
 11. The method of claim 1, wherein the conjugate is administered for multiple cycles.
 12. The method of claim 1, wherein the human has abnormal cytogenetics.
 13. The method of claim 1, wherein the conjugate is a chemical conjugate.
 14. The method of claim 1, wherein the conjugate is a recombinantly expressed protein.
 15. The method of claim 1, wherein the conjugate is expressed as a single polypeptide comprising the catalytic and translocation domains of diphtheria toxin and human IL-3.
 16. The method of claim 1, wherein the conjugate comprises amino acid residues 1 to 388 of diphtheria toxin linked via a peptide bond to human IL-3.
 17. The method of claim 1, wherein the plasmacytoid dendritic cell cancer is refractory.
 18. The method of claim 1, wherein the human is in a state of remission from the plasmacytoid dendritic cell cancer.
 19. The method of claim 1, wherein the human has been previously treated with a therapeutic agent and/or has undergone radiation therapy.
 20. The method of claim 1, wherein the human is currently being administered a therapeutic agent other than a human IL-3-diphtheria toxin conjugate and/or is undergoing radiation therapy.
 21. The method of claim 1, wherein the human has relapsed from plasmacytoid dendritic cell cancer.
 22. The method of claim 1, wherein the human has failed plasmacytoid dendritic cell cancer treatment.
 23. The method of claim 1, wherein the human is susceptible to adverse reactions from other plasmacytoid dendritic cell cancer therapies.
 24. The method of claim 1, wherein the human is refractory to chemotherapy.
 25. The method of claim 1, wherein the human has not been previously treated for plasmacytoid dendritic cell cancer. 